Novel Compound and Methods for its Production

ABSTRACT

There is provided inter alia a compound of formula (I): 
     
       
         
         
             
             
         
       
     
     for use in treatment of viral infection or as an immunosuppressant.

The present application claims priority under 35 U.S.C. §119(a) to Great Britain Patent Application No. 1105293.3, filed Mar. 29, 2011; Great Britain Patent Application No. 1113629.8, filed Aug. 8, 2011; and Great Britain Patent Application No. 1202060.8, filed Feb. 7, 2012. The entire disclosure of each of the foregoing applications is incorporated by reference herein.

Introduction

The present invention relates to a sanglifehrin analogue, that is useful both as a cyclophilin inhibitor, e.g. in the treatment of viral infection by viruses such as Hepatitis C virus (HCV), Hepatitis B Virus (HBV) and Human Immunodeficiency Virus (HIV) and/or as an immunosuppressant e.g. for use in prophylaxis of transplant rejection and as an anti-inflammatory agent, e.g. for use in inflammatory disorders. The present invention also provides methods for its use in medicine, in particular for the treatment of HCV or HIV infection and for use as an immunosuppressant or anti-inflammatory agent, in diseases where inhibition of the Mitochondrial Permeability Transition Pore (mPTP) is useful such as muscular dystrophy or as an intermediate in the generation of further medicinally useful compounds.

BACKGROUND OF THE INVENTION Hepatitis C

Hepatitis C virus (HCV) is a positive strand RNA virus, and infection is a leading cause of post-transfusional hepatitis. HCV is the most common chronic blood borne infection, and the leading cause of death from liver disease in United States. The World Health Organization estimates that there are more than 170 million chronic carriers of HCV infection, which is about 3% of the world population. Among the un-treated HCV-infected patients, about 70%-85% develop chronic HCV infection, and are therefore at high risk to develop liver cirrhosis and hepatocellular carcinoma. In developed countries, 50-76% of all cases of liver cancer and two-thirds of all liver transplants are due to chronic HCV infection (Manns et al, 2007).

In addition to liver diseases, chronically infected patients may also develop other chronic HCV-related diseases, and serve as a source of transmission to others. HCV infection causes non-liver complications such as arthralgias (joint pain), skin rash, and internal organ damage predominantly to the kidney. HCV infection represents an important global health-care burden, and currently there is no vaccine available for hepatitis C (Strader et al., 2004; Jacobson et al. 2007; Manns et al., 2007; Pawlotsky, 2005; Zeuzem & Hermann, 2002).

Treatment of HCV

The current standard of care (SoC) is subcutaneous injections of pegylated interferon-α (pIFNα) and oral dosing of the antiviral drug ribavirin for a period of 24-48 weeks. Success in treatment is defined by sustained virologic response (SVR), which is defined by absence of HCV RNA in serum at the end of treatment period and 6 months later. Overall response rates to SoC depend mainly on genotype and pretreatment HCV RNA levels. Patients with genotype 2 and 3 are more likely to respond to SoC than patients infected with genotype 1 (Melnikova, 2008; Jacobson et al., 2007).

A significant number of HCV patients do not respond adequately to the SoC treatment, or cannot tolerate the therapy due to side effects, leading to frequent issues with completion of the full course. The overall clinical SVR rate of SoC is only around 50% (Melnikova, 2008). Development of resistance is another underlying factor for failure of treatment (Jacobson et al. et al. 2007). SoC is also contraindicated in some patients who are not considered candidates for treatment, such as patients with past significant episodes of depression or cardiac disease. Side effects of the SoC, which frequently lead to discontinuation of treatment, include a flu-like illness, fever, fatigue, haematological disease, anemia, leucopaenia, thrombocytopaenia, alopecia and depression (Manns et al., 2007).

Considering the side effects associated with the lengthy treatments using SoC, development of resistance, and suboptimum overall rate of success, more efficacious and safer new treatments are urgently needed for treatment of HCV infection. The objectives of new treatments include improved potency, improved toxicity profile, improved resistance profile, improved quality of life and the resulting improvement in patient compliance. HCV has a short life cycle and therefore development of drug resistance during drug therapy is common.

Novel, specifically targeted antiviral therapy for hepatitis C(STAT-C) also known as direct acting antiviral (DAA) drugs are being developed that target viral proteins such as viral RNA polymerase NS5B or viral protease NS3 (Jacobson et al, 2007; Parfieniuk et al., 2007). In addition, novel compounds also are being developed that target human proteins (e.g. cyclophilins) rather than viral targets, which might be expected to lead to a reduction in incidence of resistance during drug therapy (Manns et al., 2007; Pockros, 2008; Pawlotsky J-M, 2005).

Cyclophilin Inhibitors

Cyclophilins (CyP) are a family of cellular proteins that display peptidyl-prolyl cis-trans isomerase activity facilitating protein conformation changes and folding. CyPs are involved in cellular processes such as transcriptional regulation, immune response, protein secretion, and mitochondrial function. HCV virus recruits CyPs for its life cycle during human infection. Originally, it was thought that CyPs stimulate the RNA binding activity of the HCV non-structural protein NS5B RNA polymerase that promotes RNA replication, although several alternative hypotheses have been proposed including a requirement for CyP PPlase activity. Various isoforms of CyPs, including A and B, are believed to be involved in the HCV life cycle (Yang et al., 2008; Appel et al., 2006; Chatterji et al., 2009; Gaither et al., 2010). The ability to generate knockouts in mice (Colgan et al., 2000) and human T cells (Braaten and Luban, 2001) indicates that CyPA is optional for cell growth and survival. Similar results have been observed with disruption of CyPA homologues in bacteria (Herrler et al., 1994), Neurospora (Tropschug et al., 1989) and Saccharomyces cerevisiae (Dolinski et al. 1997). Therefore, inhibiting CyPs represent a novel and attractive host target for treating HCV infection, and a new potential addition to current SoC or STAT-C/DAA drugs, with the aim of increasing SVR, preventing emergence of resistance and lowering treatment side effects.

Cyclosporine A (Inoue et al. 2003) (“CsA”) and its closely structurally related non-immunosuppressive clinical analogues DEBIO-025 (Paeshuyse et al. 2006; Flisiak et al. 2008), NIM811 (Mathy et al. 2008) and SCY-635 (Hopkins et al., 2009) are known to bind to cyclophilins, and as cyclophilin inhibitors have shown in vitro and clinical efficacy in the treatment of HCV infection (Crabbe et al., 2009; Flisiak et al. 2008; Mathy et al. 2008; Inoue et al., 2007; Ishii et al., 2006; Paeshuyse et al., 2006). Although earlier resistance studies on CsA showed mutations in HCV NS5B RNA polymerase and suggested that only cyclophilin B would be involved in the HCV replication process (Robida et al., 2007), recent studies have suggested an essential role for cyclophilin A in HCV replication (Chatterji et al. 2009; Yang et al., 2008). Considering that mutations in NS5A viral protein are also associated with CsA resistance and that NS5A interacts with both CyPA and CypB for their specific peptidyl-prolyl cis/trans isomerase (PPlase) activity, a role for both cyclophilins in viral life cycle is further suggested (Hanoulle et al., 2009).

The anti-HCV effect of cyclosporine analogues is independent of the immunosuppressive property, which is dependent on calcineurin. This indicated that the essential requirement for HCV activity is CyP binding and calcineurin binding is not needed. DEBIO-025, the most clinically advanced cyclophilin inhibitor for the treatment of HCV, has shown in vitro and in vivo potency against the four most prevalent HCV genotypes (genotypes 1, 2, 3, and 4). Resistance studies showed that mutations conferring resistance to DEBIO-025 were different from those reported for polymerase and protease inhibitors, and that there was no cross resistance with STAT-C/DAA resistant viral replicons. More importantly, DEBIO-025 also prevented the development of escape mutations that confer resistance to both protease and polymerase inhibitors (Crabbe et al., 2009).

However, the CsA-based cyclophilin inhibitors in clinical development have a number of issues, which are thought to be related to their shared structural class, including: certain adverse events that can lead to a withdrawal of therapy and have limited the clinical dose levels; variable pharmacokinetics that can lead to variable efficacy; and an increased risk of drug-drug interactions that can lead to dosing issues.

The most frequently occurring adverse events (AEs) in patients who received DEBIO-025 included jaundice, abdominal pain, vomiting, fatigue, and pyrexia. The most clinically important AEs were hyperbilirubinemia and reduction in platelet count (thrombocytopaenia). Peg-IFN can cause profound thrombocytopaenia and combination with DEBIO-025 could represent a significant clinical problem. Both an increase in bilirubin and decrease in platelets have also been described in early clinical studies with NIM-811 (Ke et al., 2009). Although the hyperbilirubinemia observed during DEBIO-025 clinical studies was reversed after treatment cessation, it was the cause for discontinuation of treatment in 4 out of 16 patients, and a reduction in dose levels for future trials. As the anti-viral effect of cyclophilin inhibitors in HCV is dose related, a reduction in dose has led to a reduction in anti-viral effect, and a number of later trials with CsA-based cyclophilin inhibitors have shown no or poor reductions in HCV viral load when dosed as a monotherapy (Lawitz et al., 2009; Hopkins et al., 2009; Nelson et al., 2009). DEBIO-025 and cyclosporine A are known to be inhibitors of biliary transporters such as bile salt export pumps and other hepatic transporters (especially OAT1B1/OAT1B3/MRP2/MRP3/cMOAT/ABCC2) (Crabbe et al., 2009). It has been suggested that the interaction with biliary transporters, in particular MRP2, may be the cause of the hyperbilirubinaemia seen at high dose levels of DEBIO-025 (Nelson et al., 2009, Wring et al., 2010). CsA class-related drug-drug interactions (DDIs) via inhibition of other drug transporters such as P-glycoprotein (Pgp/MDR1), BSEP, OAT1B1 and OAT1B3 (Konig et al., 2010) may also be a concern, potentially limiting certain combinations and use in some patients undergoing treatment for co-infections such as HIV (Seden et al., 2010).

Moreover, DEBIO-025 and cyclosporine A are substrates for metabolism by cytochrome P450 (especially CYP3A4), and are known to be substrates and inhibitors of human P-glycoprotein (MDR1) (Crabbe et al., 2009). Cyclosporine A has also been shown to be an inhibitor of CYP3A4 in vitro (Niwa et al., 2007). This indicates that there could be an increased risk of drug-drug interactions with other drugs that are CYP3A4 substrates, inducers or inhibitors such as for example ketoconazole, cimetidine and rifampicin. In addition, interactions are also expected with drugs that are subject to transport by P-glycoprotein (e.g. digoxin), which could cause severe drug-drug interactions in HCV patients receiving medical treatments for other concomitant diseases (Crabbe et al. 2009). CsA is also known to have highly variable pharmacokinetics, with early formulations showing oral bioavailability from 1-89% (Kapurtzak et al., 2004). Without expensive monitoring of patient blood levels, this can lead to increased prevalence of side effects due to increased plasma levels, or reduced clinical response due to lowered plasma levels.

Considering that inhibition of cyclophilins represent a promising new approach for treatment of HCV, there is a need for discovery and development of more potent and safer CyP inhibitors for use in combination therapy against HCV infection.

Sanglifehrins

Sanglifehrin A (SfA) and its natural congeners belong to a class of mixed non-ribosomal peptide/polyketides, produced by Streptomyces sp. A92-308110 (also known as DSM 9954) (see WO 97/02285), which were originally discovered on the basis of their high affinity to cyclophilin A (CyPA). SfA is the most abundant component in fermentation broths and exhibits approximately 20-fold higher affinity for CyPA compared to CsA. This has led to the suggestion that sanglifehrins could be useful for the treatment of HCV (WO2006/138507). Sanglifehrins have also been shown to exhibit a lower immunosuppressive activity than CsA when tested in vitro (Sanglier et al., 1999; Fehr et al., 1999). SfA binds with high affinity to the CsA binding site of CyPA (Kallen et al., 2005).

Biosynthesis of Sanglifehrins

Sanglifehrins are biosynthesised by a mixed polyketide synthase (PKS)/Non-ribosomal peptide synthetase (NRPS) (see WO2010/034243). The 22-membered macrolide backbone consists of a polyketide carbon chain and a tripeptide chain. The peptide chain consists of one natural amino acid, valine, and two non-natural amino acids: (S)-meta-tyrosine and (S)-piperazic acid, linked by an amide bond. Hydroxylation of phenylalanine (either in situ on the NRPS or prior to biosynthesis) to generate (S)-meta-tyrosine is thought to occur via the gene product of sfaA.

Immunosuppressive Action of Sanglifehrins

The immunosuppressive mechanism of action of SfA is different to that of other known immunophilin-binding immunosuppressive drugs such as CsA, FK506 and rapamycin. SfA does not inhibit the phosphatase activity of calcineurin, the target of CsA (Zenke et al. 2001), instead its immunosuppressive activity has been attributed to the inhibition of interleukin-6 (Hartel et al., 2005), interleukin-12 (Steinschulte et al., 2003) and inhibition of interleukin-2-dependent T cell proliferation (Zhang & Liu, 2001). However, the molecular target and mechanism through which SfA exerts its immunosuppressive effect is hitherto unknown.

The molecular structure of SfA is complex and its interaction with CyPA is thought to be mediated largely by the macrocyclic portion of the molecule. In fact, a macrocyclic compound (hydroxymacrocycle) derived from oxidative cleavage of SfA has shown strong affinity for CyPA (Sedrani et al., 2003). X-ray crystal structure data has shown that the hydroxymacrocycle binds to the same active site of CyPA as CsA. Analogues based on the macrocycle moiety of SfA have also previously been shown to be devoid of immunosuppressive properties (Sedrani et al., 2003), providing opportunity for design of non-immunosuppressive CyP inhibitors for potential use in HCV therapy.

Converse to this, there is also an opportunity to develop immunosuppressive agents with low toxicity for use in such areas as prophylaxis of transplant rejection, autoimmune, inflammatory and respiratory disorders, including, but not limited to, Crohn's disease, Behcet syndrome, uveitis, psoriasis, atopic dermatitis, rheumatoid arthritis, nephritic syndrome, aplastic anaemia, biliary cirrhosis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and celiac disease. Sanglifehrins have been shown to have a novel mechanism of immunosuppressive activity (Zenke et al., 2001), potentially acting through dendritic cell chemokines (Immecke et al., 2011), and there is therefore an opportunity to develop agents with a mechanism of action different to current clinical agents, such as cyclosporine A, rapamycin and FK506. Sanglifehrin A has been shown to be 10 fold less potent than Cyclosporine A, so the ideal novel agent would have improved potency and/or therapeutic window.

Other Therapeutic Uses of Cyclophilin Inhibitors Human Immunodeficiency Virus (HIV)

Cyclophilin inhibitors, such as CsA and DEBIO-025 have also shown potential utility in inhibition of HIV replication. The cyclophilin inhibitors are thought to interfere with function of CyPA during progression/completion of HIV reverse transcription (Ptak et al., 2008). However, when tested clinically, DEBIO-025 only reduced HIV-1 RNA levels and >1 log 10 copies/mL in nine and two patients respectively, whilst 27 of the treated patients showed no reduction in HIV-1 RNA levels (Steyn et al., 2006). Following this, DEBIO-025 was trialled in HCV/HIV coinfected patients, and showed better efficacy against HCV, and the HIV clinical trials were discontinued (see Watashi et al., 2010).

Treatment of HIV

More than 30 million people are infected by HIV-1 worldwide, with 3 million new cases each year. Treatment options have improved dramatically with the introduction of highly active antiretroviral therapy (HAART) (Schopman et al., 2010), By 2008, nearly 25 antiretroviral drugs had been licensed for treatment of HIV-1, including nine nucleoside reverse transcriptase inhibitors (NRTI), four non-nucleoside reverse transcriptase inhibitors (NNRTI), nine protease inhibitors (PI), one fusion inhibitor, one CCR5 inhibitor and one integrase inhibitor (Shafer and Schapiro, 2008). However, none of these current regimens leads to complete viral clearance, they can lead to severe side effects and antiviral resistance is still a major concern. Therefore, there still remains a need for new antiviral therapies, especially in mechanism of action classes where there are no approved drugs, such as is the case for cyclophilin inhibitors.

Hepatitis B Virus

Hepatitis B is a DNA virus of the family hepadnaviridae, and is the causative agent of Hepatitis B. As opposed to the cases with HCV and HIV, there have been very few published accounts of activity of cyclophilin inhibitors against Hepatitis B virus. Ptak et al. 2008 have described weak activity of Debio-025 against HBV (IC50 of 4.1 μM), whilst Xie et al., 2007 described some activity of CsA against HBV (IC50>1.3 μg/mL). This is in contrast to HIV and HCV, where there are numerous reports of nanomolar antiviral activity of cyclophilin inhibitors.

Treatment of HBV

HBV infects up to 400 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma. As of 2008, there were six drugs licensed for the treatment of HBV; interferon alpha and pegylated interferon alpha, three nucleoside analogues (lamivudine, entecavir and telbivudine) and one nucleotide analogue (adefovir dipivoxil). However, due to high rates of resistance, poor tolerability and possible side effects, new therapeutic options are needed (Ferir et al., 2008).

Inhibition of the Mitochondrial Permeability Transition Pore (mPTP)

Opening of the high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). This is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca2+ toxicity, and ischaemia/reperfusion. Inhibition of Cyclophilin D (also known as Cyclophilin F) by cyclophilin inhibitors has been shown to block opening of permeability transition pores and protects cell death after these stresses. Cyclophilin D inhibitors may therefore be useful in indications where the mPTP opening has been implicated, such as muscular dystrophy, in particular Ullrich congenital muscular dystrophy and Bethlem myopathy (Millay et al., 2008, WO2008/084368, Palma et al., 2009), multiple sclerosis (Forte et al., 2009), diabetes (Fujimoto et al., 2010), amyotrophic lateral sclerosis (Martin 2009), bipolar disorder (Kubota et al., 2010), Alzheimer's disease (Du and Yan, 2010), Huntington's disease (Perry et al., 2010), recovery after myocardial infarction (Gomez et al., 2007) and chronic alcohol consumption (King et al., 2010).

Further Therapeutic Uses

Cyclophilin inhibitors have potential activity against and therefore in the treatment of infections of other viruses, such as Varicella-zoster virus (Ptak et al., 2008), Influenza A virus (Liu et al., 2009), Severe acute respiratory syndrome coronavirus and other human and feline coronaviruses (Chen et al., 2005, Ptak et al., 2008), Dengue virus (Kaul et al., 2009), Yellow fever virus (Qing et al., 2009), West Nile virus (Qing et al., 2009), Western equine encephalitis virus (Qing et al., 2009), Cytomegalovirus (Kawasaki et al., 2007) and Vaccinia virus (Castro et al., 2003).

There are also reports of utility of cyclophilin inhibitors and cyclophilin inhibition in other therapeutic areas, such as in cancer (Han et al., 2009).

General Comments on Sanglifehrins

One of the issues in drug development of compounds such as sanglifehrins is rapid metabolism and glucuronidation, leading to low oral bioavailability. This can lead to an increased chance of food effect, more frequent incomplete release from the dosage form and higher interpatient variability.

Therefore there remains a need to identify novel cyclophilin inhibitors, which may have utility, particularly in the treatment of HCV infection, but also in the treatment of other disease areas where inhibition of cyclophilins may be useful, such as HIV infection, Muscular Dystrophy or aiding recovery after myocardial infarction or where immunosuppression or anti-inflammatory effect is useful. Preferably, such cyclophilin inhibitors have improved properties over the currently available cyclophilin inhibitors, including one or more of the following properties: longer half-life or increased oral bioavailability, possibly via reduced P450 metabolism and/or reduced glucuronidation, improved water solubility, improved potency against HCV, reduced toxicity (including hepatotoxicity), improved pharmacological profile, such as high exposure to target organ (e.g. liver in the case of HCV) and/or long half life (enabling less frequent dosing), reduced drug-drug interactions, such as via reduced levels of CYP3A4 metabolism and inhibition and reduced (Pgp) inhibition (enabling easier multi-drug combinations) and improved side-effect profile, such as low binding to MRP2, leading to a reduced chance of hyperbilirubinaemia, lower immunosuppressive effect, improved activity against resistant virus species, in particular CsA and CsA analogue (e.g. DEBIO-025) resistant virus species and higher therapeutic (and/or selectivity) index. The present invention discloses a novel sanglifehrin analogue which may have one or more of the above properties. In particular, the present invention discloses a novel mutasynthetic sanglifehrin analogue, which is anticipated to have reduced metabolism via P450 or glucuronidation, for example as shown by increased microsome half-life and/or reduced improved potency against HCV, for example as shown by a low replicon EC₅₀.

In addition, there is also a need to develop a novel immunosuppressive agent, which may have utility in the prophylaxis of transplant rejection, or in the treatment of autoimmune, inflammatory and respiratory disorders. Preferably, such an immunosuppressant will have improved properties over the known natural sanglifehrins, including one or more of the following properties: longer half-life or increased oral bioavailability, possibly via reduced P450 metabolism and/or reduced glucuronidation, improved water solubility, improved potency in immunosuppressive activity, such as might be seen in T-cell proliferation assays, reduced toxicity (including hepatotoxicity), improved pharmacological profile, such as high exposure to target organ and/or long half-life (enabling less frequent dosing), reduced drug-drug interactions, such as via reduced levels of CYP3A4 metabolism and inhibition and reduced (Pgp) inhibition (enabling easier multi-drug combinations) and improved side-effect profile. The present invention discloses a novel sanglifehrin analogue which may have one or more of the above properties. In particular, the present invention discloses a novel derivative, which has reduced metabolism via P450 or glucuronidation, for example as shown by increased microsome half-life and/or improved immunosuppressive potency, for example as shown by a low t-cell proliferation IC₅₀.

Thus, as can be seen from the Examples, the compound of the invention has the following favourable therapeutically relevant properties:

-   -   improved antiviral potency against HCV and HIV as compared with         the prior art cyclophilin inhibitors Cyclosporin A, DEBIO-025         (alisporivir) and Sanglifehrin A;     -   reduced clearance and increased oral expose as compared with the         prior art compound Sanglifehrin A;     -   more potent inhibition of CypA PPlase activity as compared with         the prior art cyclophilin inhibitors Cyclosporin A, DEBIO-025         (alisporivir) and Sanglifehrin A;     -   improved side effect profile and reduced drug-drug interactions         as demonstrated by reduced inhibition of bilirubin transporters         (OATP-1B1, OATP-1B3, MRP2 and MRP3) and reduced inhibition of         xenobiotic transporters (Pgp and BSEP).

SUMMARY OF THE INVENTION

The present invention provides a novel macrocyclic sanglifehrin analogue, which has been generated by semisynthetic modification of mutasynthetic sanglifehrins. This analogue may be generated by dihydroxylation of a mutasynthetic sanglifehrin, such as described in formula IIA and formula IIB, followed by cleavage to generate the aldehydic macrocycle, followed by further chemistry, including Horner-Emmons type reactions and other coupling reactions involving an aldehyde. As a result, the present invention provides a macrocyclic sanglifehrin analogue, methods for the preparation of this compound, and methods for the use of this compound in medicine or as an intermediate in the production of further compounds.

Therefore, in a first aspect, the present invention provides a macrocyclic sanglifehrin analogue according to formula (I) below, or a pharmaceutically acceptable salt thereof:

including any tautomer thereof; and including a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl group and methanol.

The above structure shows a representative tautomer and the invention embraces all tautomers of the compound of formula (I) for example a keto compound where an enol compound is illustrated and vice versa.

Specific tautomers that are included within the definition of formula (I) are those in which (i) the C-53 keto group forms a hemiketal with the C-15 hydroxyl, or (ii) the C-15 and C-17 hydroxyl can combine with the C-53 keto to form a ketal. All numberings use the system for the parent sanglifehrin A structure.

The compound of formula (I), or a pharmaceutically acceptable salt thereof, may optionally be present in the form of a pharmaceutically acceptable solvate, such as a hydrate.

In a further aspect, the present invention provides a macrocyclic sanglifehrin analogue according to formula (I) in solid crystalline form (Form I).

DEFINITIONS

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example “an analogue” means one analogue or more than one analogue.

As used herein the term “analogue(s)” refers to chemical compounds that are structurally similar to another but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group).

As used herein the term “sanglifehrin(s)” refers to chemical compounds that are structurally similar to sanglifehrin A but which differ slightly in composition (as in the replacement of one atom by another or in the presence or absence of a particular functional group), in particular those generated by fermentation of Streptomyces sp. A92-308110. Examples include the sanglifehrin-like compounds discussed in WO97/02285 and WO98/07743, such as sanglifehrin B.

As used herein the term “mutasynthetic sanglifehrin(s)” or “mutasynthetic sanglifehrin analogue(s)” refers to chemical compounds that are structurally similar to sanglifehrin A, B, C or D but which differ slightly in composition (as in the replacement of one or more atom by another or in the presence or absence of a particular functional group), in particular, those generated by fermentation of Streptomyces sp. A92-308110 or a mutant thereof, where the culture is fed with a meta-tyrosine analogue.

As used herein the term “meta-tyrosine analogue(s)” refers to chemical compounds that are structurally similar to meta-tyrosine but which differ slightly in composition (as in the replacement of one or more atom by another or in the presence or absence of a particular functional group), in particular, those described in formula (III).

As used herein, the term “macrocyclic analogue”, “macrocyclic sanglifehrin analogue” or “macrocyclic sanglifehrin”, refers to a compound referred to above as representing the invention in its broadest aspect, for example a compound according to formula (I) above, or a pharmaceutically acceptable salt thereof. These compounds are also referred to as “compounds of the invention” or “derivatives of sanglifehrin” or “sanglifehrin analogues” and these terms are used interchangeably in the present application.

As used herein, the term “HCV” refers to Hepatitis C Virus, a single stranded, RNA, enveloped virus in the viral family Flaviviridae.

As used herein, the term “HIV” refers to Human Immunodeficiency Virus, the causative agent of Human Acquired Immune Deficiency Syndrome.

As used herein, the term “bioavailability” refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc. Various tests for bioavailability that would be familiar to a person of skill in the art are described herein (see also Egorin et al. 2002).

The term “water solubility” as used in this application refers to solubility in aqueous media, e.g. phosphate buffered saline (PBS) at pH 7.4, or in 5% glucose solution. Tests for water solubility are given below in the Examples as “water solubility assay”.

The pharmaceutically acceptable salts of compounds of the invention such as the compound of formula (I) include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Hydrochloric acid salts are of particular interest. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. References hereinafter to a compound according to the invention include both a compound of formula (I) and its pharmaceutically acceptable salts.

As used herein, the term “alkyl” represents a straight chain or branched alkyl group, containing typically 1-10 carbon atoms, for example a C₁₋₆ alkyl group. Examples of alkyl groups include C₁₋₄ alkyl groups such as methyl, ethyl, n-propyl, i-propyl, and n-butyl.

The term “treatment” includes prophylactic as well as therapeutic treatment.

The term “formula II” refers to formula IIA and formula IIB collectively.

FIGURE LEGEND

FIG. 1: ¹H NMR of compound 24

FIG. 2: X-ray powder diffraction pattern of compound 24 in solid crystalline form (Form I)

DESCRIPTION OF THE INVENTION

The present invention provides a macrocyclic sanglifehrin analogue, as set out above, methods for preparation of this compound and methods for the use of this compound in medicine.

In one embodiment, the compound is a methanol adduct thereof in which a hemi-ketal is formed by the combination of the C-53 keto and the C-15 hydroxyl groups and methanol. In another embodiment it is not.

In an embodiment of the invention, the double bond at the C26, 27 position is in the cis form, as represented by the following formula:

Such a compound may be produced during chemical synthesis.

In a further embodiment, there is provided a macrocyclic sanglifehrin analogue according to formula (I) in solid crystalline form. In particular, there is provided a solid crystalline form (Form I) of a macrocyclic sanglifehrin analogue according to formula (I) which is obtainable (or obtained) by crystallization of amorphous macrocyclic sanglifehrin analogue according to formula (I) from methyl isobutyl ketone (MIBK). In one embodiment, said amorphous form is slurried in MIBK and the temperature is cycled between a minimum and maximum temperature for a total period of time of, for example, 1 hour, 2 hours, 5 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days 7 days or 2 weeks. In one embodiment, the temperature is cycled between ambient temperature and 60° C., for example between ambient temperature and 40° C. In one embodiment, the temperature cycles between the minimum and maximum temperature (and visa versa) every 2-8 hours, for example, every 3-5 hours or every 4 hours. In a preferred embodiment, the temperature is cycled between ambient temperature and 40° C. every 4 hours for a total of 5 days.

A method of crystallization of the amorphous form of macrocyclic sanglifehrin analogue according to formula (I) is described in detail in Example 8.

A macrocyclic sanglifehrin analogue according to formula (I) in the form of crystalline polymorph Form I has an X-ray powder diffraction (XRPD) pattern substantially as shown in FIG. 2. Table 2 (of Example 8) shows the peak listings and relative intensities. The method of obtaining the XRPD data is described in the General Methods.

Thus, there is provided a macrocyclic sanglifehrin analogue according to formula (I) in a crystalline form (Form I) having an XRPD pattern with at least at least one (for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or all seventeen) signals at 8.3, 8.5, 11.1, 12.6, 13.9, 14.3, 15.0, 16.9, 17.7, 18.6, 19.0, 20.1, 20.5, 20.9, 21.2, 21.7 and 23.0 (±0.2 degrees, 2-theta values), which signals constitute the major signals in the XRPD pattern of the Form I polymorph. The signals at 8.3, 8.5, 11.1, 13.9, 17.7, 18.6, 19.0, 20.5, 20.9 and 23.0 degrees 2-theta have comparatively high relative intensity (more than 26%—see FIG. 2) and therefore it is preferred to see at least one (for example, one, two, three, four, five, six, seven, eight, nine or all ten) of these. The signals at 13.9, 17.7, 19.0, 20.5 and 23.0 degrees 2-theta have particularly high relative intensity (more than 50%—see FIG. 2) and therefore it is preferred to see at least one (for example, one, two, three, four, or all five) of these.

The term “relative intensity” will be understood to mean the intensity given as a percentage as the intensity of the signal of highest intensity in the spectrum (which corresponds to the peak at 13.9 degrees 2-theta), as illustrated by FIG. 2.

In general, the compound of the invention is prepared by mutasynthesis to generate compounds of formula (II), followed by semisynthesis.

In general, a process for preparing precursors of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises:

-   -   Inoculating a fermentation broth with a culture of a         sanglifehrin producer (such as Streptomyces sp. A92-308110, also         known as DSM 9954) or more preferably, a sanglifehrin producer         with the sfaA gene or sfaA gene homologue inactivated or         deleted;     -   Feeding the fermentation broth with a meta-tyrosine analogue (as         shown in formula (III), for example (S)-methyl         2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate,         DL-5-fluoro-meta-tyrosine (9) or methyl         2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10))     -   Allowing fermentation to continue until compounds of formula IIA         and formula IIB are produced     -   Extracting and isolating compounds of formula IIA and formula         IIB     -   Semisynthetic derivatisation of compounds of formula IIA and         formula IIB to generate the compound of formula I.

Compounds of formula (III) are defined as follows:

where R₁₀ represents H or an ester forming group such as an alkyl group, e.g. C₁₋₆alkyl such as Me.

The feed may be racemic or the L-form of a compound of formula (III).

Compounds of formula (III) are either commercially available or prepared by standard organic synthetic chemistry techniques. One generic route to compounds of formula (III) is as shown in the following scheme 1a.

Scheme 1a: a) coupling aldehyde of formula (IV) with suitable fragment, e.g. (R₁₁O)₂P(O)CH(NHPG)CO₂R₁₀, and b) hydrogenation and deprotection as necessary. PG=protecting group.

Aldehydes of formula (IV) may be commercially available or readily synthesised by one skilled in the art. Protection and deprotection chemistry may need to be employed in generating compounds of formula (III) from compounds of formula (IV). These techniques are known to one skilled in the art and suitable protecting groups are described in Greene's Protective Groups in Organic Synthesis (Wuts and Greene, 4^(th) Edition, 2007)

Following generation of compounds of formula (IIA) and formula (IIB), the compounds of the invention are prepared by semi-synthetic derivatisation. Semisynthetic methods for generating the sanglifehrin macrocyclic aldehyde are described in U.S. Pat. No. 6,124,453, Metternich et al., 1999, Banteli et al., 2001 and Sedrani et al., 2003.

In general, the semisynthetic process for preparing certain compounds of formula (I) or a pharmaceutically acceptable salt thereof from a sanglifehrin mutasynthetic analogue comprises:

-   -   (a) dihydroxylation of the sanglifehrin analogue;     -   (b) oxidative cleavage of the 1,2-diol to yield an aldehyde; and     -   (c) coupling said aldehyde with a stabilised carbanion (or         canonical form thereof), such as a phosphonate carbanion, using         a compound of formula V.

This is shown retrosynthetically below:

Wherein for sanglifehrin A mutasynthetic analogues,

-   -   R₁₁ groups, which may be the same or different, independently         represent alkyl (e.g. C1-4alkyl) or benzyl.     -   Hence, a process for preparing a compound of the invention         comprises reacting a compound of formula (V) with an aldehydic         macrocycle (compound of formula (VI)).

The preparation of compounds of formula (VI) may be performed by a process analogous to that described previously for the conversion of sanglifehrin A to its corresponding aldehydic macrocycle (Metternich et al. 1999). Briefly, the compound of formula (II) is dihydroxylated using modified Sharpless conditions (catalytic osmium tetroxide). The use of the chiral ligands aids in promoting selectivity. The resultant diol can then be cleaved oxidatively, using for instance sodium periodate. The resultant compound of formula VI can then be used as a substrate for derivatisation to a homologated amide, ester or ketone. Typically a compound of formula (V) is dissolved in an aprotic solvent, cooled and the treated with a base, for example sodium hydride. A compound of formula (VI) is then added and the reaction warmed in temperature. After a suitable period of time the reaction is stopped and the compound of formula I is purified by standard conditions (e.g. preparative HPLC, preparative TLC etc, normal phase flash chromatography).

Compounds of formula (V) may be known or may be prepared using known methods.

As shown in scheme 1 (below) the appropriate amine may be used to treat chloroacetyl chloride or similar to form an alpha-chloroamide. The alpha-chloroamide is then treated in an Arbuzov reaction to generate a compound of formula V. Other routes to compounds of formula V will be apparent to one skilled in the art.

Further compounds of formula (V) may be known or readily synthesised from available carboxylic acid derivatives (e.g. R₃COX) wherein R₃ is the 1,2-oxazinane ring shown in scheme 2. As shown in scheme 2 (below) the carboxylic acid derivative may be coupled onto a methyl phosphonate after the phosphonate has been treated with base. This yields a compound of formula (V), though other routes to compounds of formula V will be apparent to one skilled in the art.

If desired or necessary, protecting groups may be employed to protect functionality in the aldehydic macrocycle or macrocycle, or in compounds of formula V as described in T. W. Green, P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley-Interscience, New York, 1999.

In addition to the specific methods and references provided herein a person of skill in the art may also consult standard textbook references for synthetic methods, including, but not limited to Vogel's Textbook of Practical Organic Chemistry (Furniss et al., 1989) and March's Advanced Organic Chemistry (Smith and March, 2001).

A sanglifehrin analogue according to the invention may be administered alone or in combination with other therapeutic agents. Co-administration of two (or more) agents may allow for lower doses of each to be used, thereby reducing side effect, can lead to improved potency and therefore higher SVR, and a reduction in resistance.

Therefore in one embodiment, the mutasynthetic sanglifehrin analogue is co-administered with one or more therapeutic agent/s for the treatment of HCV infection, taken from the standard of care treatments. This could be an interferon (e.g. pIFNα and/or ribavirin).

In an alternative embodiment, a sanglifehrin macrocycle of the invention is co-administered with one or more other anti-viral agents, such as a STAT-C (specifically targeted agent for treatment of HCV) or DAA (direct acting antivirals), which could be one or more of the following: Non-nucleoside Polymerase inhibitors (e.g. ABT-333, ABT-072, BMS 791325, IDX375, VCH-222, BI 207127, ANA598, VCH-916, GS 9190, PF-00868554 (Filibuvir) or VX-759), Nucleoside or nucleotide polymerase inhibitors (e.g. 2′-C-methylcytidine, 2′-C-methyladenosine, R1479, PSI-6130, R7128, R1626, PSI 7977 or IDX 184), Protease inhibitors (e.g. ABT-450, ACH-1625, BI 201355, BILN-2061, BMS-650032, CTS1027, Danoprevir, GS 9256, GS 9451, MK 5172, IDX 320, VX-950(Telaprevir), SCH503034(Boceprevir), TMC435350, MK-7009 (Vaneprivir), R7227/ITMN-191, EA-058, EA-063 or VX 985), NS5A inhibitors (e.g. A-831, BMS 790052, BMS 824393, CY-102 or PPI-461), silymarin, NS4b inhibitors, serine C-palmitoyltransferase inhibitors, Nitazoxanide or viral entry inhibitors (e.g. PRO 206).

In an alternative embodiment, a sanglifehrin macrocycle of the invention is co-administered with one or more other anti-viral agents (such as highly active antiretroviral therapy (HAART)) for the treatment of HIV, which could be one or more of the following: nucleoside reverse transcriptase inhibitors (NRTI) (e.g. Emtricitabine or Tenofovir), non-nucleoside reverse transcriptase inhibitors (NNRTI) (e.g. Rilipivirine or Efavirenz), protease inhibitors (PI) (e.g. Ritonavir or Lopinavir), fusion inhibitors (e.g. Maraviroc or Enfuvirtide), CCR5 inhibitors (e.g. Aplaviroc or Vicriviroc), maturation inhibitors (e.g. Bevirimat), CD4 monoclonal antibodies (e.g. Ibalizumab) and integrase inhibitors (e.g. Eltiegravir).

In an alternative embodiment, a sanglifehrin macrocycle of the invention is co-administered with one or more other anti-viral agents for the treatment of HBV, which could be one or more of the following: interferons (e.g. interferon alpha or pegylated interferon alpha), nucleoside or nucleotide analogues (e.g. lamivudine, entecavir, adefovir dipivoxil or telbivudine), other immunomodulators (e.g. Thymosin alpha, CYT107 or DV-601) or HMG CoA reductase inhibitors (e.g. Simvastatin).

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compounds of the invention will normally be administered orally in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

For example, the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Advantageously, agents such as preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.

The dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration. The appropriate dosage can be readily determined by a person skilled in the art.

The compositions may contain from 0.1% by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.

It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.

Further aspects of the invention include:

-   -   A compound according to the invention for use as a         pharmaceutical;     -   A compound according to the invention for use as a         pharmaceutical for the treatment of viral infections (especially         RNA virus infections) such as HCV or HIV infection, for use as         an anti-inflammatory or for prophylaxis of organ transplant         rejection;     -   A pharmaceutical composition comprising a compound according to         the invention together with a pharmaceutically acceptable         diluent or carrier;     -   A pharmaceutical composition comprising a compound according to         the invention together with a pharmaceutically acceptable         diluent or carrier further comprising a second or subsequent         active ingredient, especially an active ingredient indicated for         the treatment of viral infections such as HCV or HIV infection,         for use as an anti-inflammatory or for prophylaxis of organ         transplant rejection;     -   A method of treatment of viral infections (especially RNA virus         infections) such as HCV or HIV infection, for use as an         anti-inflammatory or for prophylaxis of organ transplant         rejection which comprises administering to a subject a         therapeutically effective amount of a compound according to the         invention;     -   Use of a compound according to the invention for the manufacture         of a medicament for the treatment of viral infections such as         HCV or HIV infection, for use as an anti-inflammatory or for         prophylaxis of organ transplant rejection.

General Methods Materials and Methods Bacterial Strains and Growth Conditions

The sanglifehrin producer Streptomyces sp. A92-308110 (DSM no 9954, purchased from DSMZ, Braunschweig, Germany) also termed BIOT-4253 and BIOT-4370 or its derivatives, such as BIOT-4585 are maintained on medium oatmeal agar, MAM, ISP4 or ISP2 (see below) at 28° C.

BIOT-4585 (for construction methodology, see Example 1) was grown on oatmeal agar at 28° C. for 7-10 days. Spores from the surface of the agar plate were collected into 20% w/v sterile glycerol in distilled and stored in 0.5-ml aliquots at −80° C. Frozen spore stock was used for inoculating seed media SGS or SM25-3. The inoculated seed medium was incubated with shaking between 200 and 300 rpm at 5.0 or 2.5 cm throw at 27° C. for 24 hours. The fermentation medium SGP-2 or BT6 were inoculated with 2.5%-10% of the seed culture and incubated with shaking between 200 and 300 rpm with a 5 or 2.5 cm throw at 24° C. for 4-5 days. The culture was then harvested for extraction.

Meta-Tyrosine Analogue

(S)-methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate was purchased from NetChem (USA). (3-bromo-5-fluoroanisole (9-1) was purchased from Accela ChemBio Co., Ltd., (Shanghai, China) and can also be purchased from Amfinecom Inc (USA) or Apollo Scientific Ltd. (UK)). DL-5-fluoro-meta-tyrosine (9) and methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10)) were synthesised as follows.

DL-5-fluoro-meta-tyrosine (9) and methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10)

To a solution of 9-1 (20 g, 97.55 mmol) in tetrahydrofuran (100 mL) was added dropwise n-butyl lithium (43 mL, 2.5 M, 107.3 mmol) at −78° C. It was stirred for 30 minutes and N,N-dimethylformamide (15.1 mL, 195.1 mmol) was added at this temperature. It was stirred for another 30 minutes and the cold bath was removed. After 1 hour, the reaction was quenched with saturated aqueous ammonium chloride. The organic layer was washed with water and saturated aqueous sodium chloride, dried (sodium sulfate), filtered and concentrated. The residue was purified by chromatography on silica to give 9-2.

To a solution of 9-2 (6 g, 38.9 mmol) in dry DCM (200 mL) was added dropwise BBr₃ (4 M in DCM, 30 ml, 116.8 mmol) at −70° C. After the addition, the reaction mixture was stirred at −20° C. for 3 hours, ice-water was added carefully, and extracted with DCM. The organic layers were washed with water and brine, dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatographed on silica to give the desired compound 9-3.

To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate (4.64 g, 14 mmol) in DCM (150 mL) was added DBU (4.26 g, 28 mmol) at room temperature. After 10 min, 9-3 (1.95 g, 14 mmol) was added and the resulting mixture was stirred at room temperature overnight. The solution was diluted with EtOAc (150 mL), separated and the organic layer was washed with 1 N HCl, dried over Na₂SO₄, filtered and concentrated. The residue was purified by flash chromatography on silica to give 9-4.

A solution of 9-4 (1 g) in MeOH (20 mL) was hydrogenated over 200 mg of 10% Pd/C at normal pressure overnight. After removal of the catalyst by filtration, the solvent was evaporated to give 10.

To a solution of 10 (300 mg, 1.4 mmol) in EtOH (30 mL) was added aq. NaOH (2 N, 4 mL), the reaction was stirred at room temperature for 30 minutes. The solvent was removed and the residue was neutralized to pH=6 with 2 N HCl and the white crystals that formed were collected by filtration to give the target compound 9.

Alternative route to methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10)

(3,5-Difluorobromobenzene (9a-1) was purchased from Darui Fine Chemicals Co., Ltd., (Shanghai, China) and can also be purchased from Alfa Aesar or Sigma Aldrich.)

Preparation of 9a-2

To a solution of BnOH (1.61 mL, 15.54 mmol) in DMF (30 mL) was added NaH (622 mg, 60% dispersion in mineral oil, 15.54 mmol) at 0° C. Stirring was continued at room temperature for 0.5 h to give a clear solution. 9a-1 (1.79 mL, 15.54 mmol) was added at such a rate to maintain the temperature below 40° C. The mixture was stirred at room temperature overnight to give a yellow solution. The reaction was quenched by water and extracted with petroleum ether (35 mL×4). The combined organic layers were concentrated. And the residue was purified by silica gel chromatography eluting with petroleum ether to afford 9a-2 (2.544 g) as colorless oil.

Preparation of 9a-3

To a dry three flask were added Mg (170.1 mg, 7.10 mmol), anhydrous THF (10 mL), and a small quantity of iodine under nitrogen. ⅓ of 9a-2 (1.664 g, 5.9192 mmol) in THF (2 mL) was added. The mixture was heated to reflux. During this time, the yellow mixture gradually became bright yellow. Then the remaining ⅔ of 9a-2 was added dropwise, and the reaction mixture was refluxed for another 0.5 h.

To the above mixture was added DMF (0.504 mL, 6.51 mmol) slowly at 0° C. Stirring was continued for 0.5 h at room temperature. HCl (2 M, 10 mL) was added, and THF was evaporated. The residue was extracted with ethyl acetate (25 mL×3). And the combined organic layers were washed with brine and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with petroleum ether to petroleum ether/ethyl acetate=20/1 to give 9a-3 (694 mg) as colorless oil.

Preparation of 9a-5

To a solution of methyl 2-(benzyloxycarbonylamino)-2-(dimethoxyphosphoryl)acetate, 9a-4 (993 mg, 3.00 mmol) in DCM (30 mL) was added DBU (832 uL, 5.57 mmol) at room temperature. After 10 min, 9a-3 (694 mg, 3.01 mmol) was added and the resulting mixture was stirred at room temperature for 1 hr. The solution was washed with HCl (1 M, 10 mL), and the combined organic layers were dried and concentrated in vacuo. The residue was purified by flash chromatography on silica (eluting with dichloromethane/ethyl acetate=10/1) to give 9a-5 (1.11 g).

Preparation of 10

A solution of 9a-5 (100 mg) in MeOH (50 mL) was hydrogenated over 20 mg of 10% Pd/C at normal pressure for 2 hrs. After removal of the catalyst by filtration, the solvent was evaporated to give 10 (33 mg).

Media Recipes Water Used for Preparing Media was Prepared Using Millipore Elix Analytical Grade Water Purification System

SGS Seed Medium Ingredient (and supplier) Recipe Glucose (Sigma, G7021) 7.50 g Glycerol (Fisher scientific, G/0650/25) 7.50 g yeast extract (Becton Dickinson, 212770) 1.35 g malt extract (Becton Dickinson, 218630) 3.75 g potato starch (soluble) (Signma, S2004) 7.50 g NZ-amine A (Sigma, C0626) 2.50 g toasted soy flour, Nutrisoy (ADM, 063- 2.50 g 160) L-asparagine (Sigma, A0884) 1.00 g CaCO₃ (Calcitec, V/40S) 0.05 g NaCl (Fisher scientific, S/3160/65) 0.05 g KH₂PO₄ (Sigma, P3786) 0.25 g K₂HPO₄ (Sigma, P5379) 0.50 g MgSO₄•7H₂O (Sigma, M7774) 0.10 g trace element solution B 1.00 mL agar 1.00 g SAG471 Antifoam (GE Silicones, *0.20 mL SAG471) RO H₂O to final vol. of **1.00 L pre-sterilisation pH was adjusted to pH 7.0 with 10M NaOH/10M H₂SO₄ sterilised by heating 121° C., 20-30 min (autoclaving) Notes *antifoam only used in seed fermenters, NOT seed flasks **final volume adjusted accordingly to account for seed volume

Trace Element Solution B Ingredient Recipe FeSO₄•7H₂O (Sigma, F8633) 5.00 g ZnSO₄•7H₂O (Sigma, Z0251) 4.00 g MnCl₂•4H₂O (Sigma, M8530) 2.00 g CuSO₄•5H₂O (Aldrich, 20,919-8) 0.20 g (NH₄)₆Mo₇O₂₄ (Fisher scientific, 0.20 g A/5720/48) CoCl₂•6H₂O (Sigma, C2644) 0.10 g H₃BO₃ (Sigma, B6768) 0.10 g KI (Alfa Aesar, A12704) 0.05 g H₂SO₄ (95%) (Fluka, 84720) 1.00 mL RO H₂O to final vol. of 1.00 L

SGP2 Production Medium Ingredient Recipe toasted soy flour (Nutrisoy) (ADM, 063-160) 20.00 g Glycerol (Fisher scientific, G/0650/25) 40.00 g MES buffer (Acros, 172595000) 19.52 g SAG471 Antifoam (GE Silicones, SAG471) *0.20 mL RO H₂O to final vol. of **1.00 L pre-sterilisation pH adjusted to pH 6.8 with 10M NaOH sterilised by heating 121° C., 20-30 min (autoclaving) Notes *final volume adjusted accordingly to account for seed volume **antifoam was used only in fermentors not flasks

SM25-3 Medium (also termed SM25) Ingredient Glycerol (Fisher scientific, G/0650/25) 40 g Soy Peptone A3 SC (Organotechnie) 10 g Malt extract (Difco) 21 g to final vol. of 1 L pre-sterilisation pH not adjusted (i.e. pH 7.0)

Ingredient ISP4 Medium Soluble Starch (Difco) 10 g K2HPO4 1 g MgSO4•7H2O 1 g NaCl 1 g (NH4)2SO4 2 g CaCO3 2 g ISP Trace Salts Solution 1 mL Agar 20 g to final vol. of 1 L Make a paste with the starch in a small volume of cold water and bring to volume of 500 ml Add other ingredients to solution II in 500 mls water pH should be between pH 7.0 and pH 7.4 (pH 7.3) Mix two solutions together and add agar ISP Trace Salts FeSO4•7H2O 1 g MnCl2•4H2O 1 g ZnSO4•7H2O 1 g to final vol. of 1 L Store at 4 degrees C

Oatmeal Agar (ISP3) Ingredient Recipe Oatmeal 20.00 g ISP trace element solution 1.00 mL Bacto Agar (Becton Dickinson) 18.00 g RO H₂O to final vol. of 1.00 L 20 g oatmeal is cooked in 1 L water on a hotplate (or microwave) for 20 minutes. The cooked mixture is filtered through muslin/cheesecloth and brought to pH 7.2 and remade up to 1 L. 1 ml ISP trace elements solution is added. 18 g per L agar is then added before sterilizing.

MAM Agar Ingredient Recipe Wheat starch (Sigma) 10.00 g Corn steep powder (Roquette) 2.50 g Yeast extract (Becton Dickinson) 3.00 g CaCO₃ (Calcitec) 3.00 g FeSO₄ (Sigma) 0.300 g Bacto Agar (Becton 20.00 g Dickinson) RO H₂O to final vol. of 1.00 L pH 5.8 prior to autoclaving

BT6 production media Ingredient Recipe Glucose (Sigma) 50.00 g Nutrisoy (ADM) 30.00 g NaCl (Fisher) 5.00 g (NH₄)₂SO₄ (Sigma) 3.00 g CaCO₃ (Calcitec) 6.00 g RO H₂O to final vol. of 1.00 L Adjust pH to 7.0 then add CaCO₃

ISP2 agar Ingredient Recipe Yeast extract (Becton Dickinson) 4.00 g Malt Extract (Becton Dickinson) 10.0 g Dextrose (Sigma) 4.00 g Bacto Agar (Becton Dickinson) 20.0 g RO H₂O to final vol. of 1.00 L Adjust pH to 7.3 prior to adding agar and sterilizing.

General Fermentation Method

Cryopreserved spore stocks of BIOT-4585 (for construction methodology, see Example 1) were thawed at room temperature. Vegetative cultures (seed cultures) were prepared by transferring 4.0 mL of spore stock into 400 mL medium SM25 in 2 L Erlenmeyer flasks with foam plug. Cultivation was carried out for 48 hours at 27° C. and 250 rpm (5.0 cm throw). From the seed culture 25 mL was transferred into 250 mL production medium SGP2+5% HP20 in 2 L Erlenmeyer flasks with foam plug. After 24 hours cultivation at 24° C. and 250 rpm (2.5 cm throw), 2 mL of a 250 mM racemic or 125 mM enantiomerically pure solution of the desired precursor (e.g. (S)-methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate, DL-5-fluoro-meta-tyrosine (9) or methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate (10)), in 1M hydrochloric acid and 2 mL of a 250 mM methanolic solution of DL-piperazic acid was added to each production flask to give a final 1 mM concentration of the individual enantiomers of the precursors. DMSO may optionally be used in place of 1M hydrochloric acid. The DL-piperazic acid may optionally be omitted. Cultivation was continued for further four days at 24° C. and 250 rpm (2.5 cm throw).

Analysis of Culture Broths by LC-UV and LC-UV-MS

Culture broth (1 mL) and ethyl acetate (1 mL) is added and mixed for 15-30 min followed by centrifugation for 10 min. 0.4 mL of the organic layer is collected, evaporated to dryness and then re-dissolved in 0.20 mL of acetonitrile.

HPLC Conditions:

C18 Hyperclone BDS C18 Column 3u, 4.6 mm×150 mm Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJ0-4282) Column temp at 50° C. Flow rate 1 mL/min

Monitor UV at 240 nm

Inject 20 uL aliquot Solvent gradient:

0 min: 55% B 1.0 min: 55% B 6.5 min: 100% B 10.0 min: 100% B 10.05 min: 55% B 13.0 min: 55% B Solvent A is Water+0.1% Formic Acid Solvent B is Acetonitrile+0.1% Formic Acid

Under these conditions SfA elutes at 5.5 min Under these conditions SfB elutes at 6.5 min LCMS is performed on an integrated Agilent HP1100 HPLC system in combination with a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive ion mode using the chromatography and solvents described above.

QC LC-MS Method

HPLC Conditions:

C18 Hyperclone BDS C18 Column 3u, 4.6 mm×150 mm Fitted with a Phenomenex Analytical C18 Security Guard Cartridge (KJ0-4282) Column temp at 50° C. Flow rate 1 mL/min

Monitor UV at 210, 240 and 254 nm

Solvent gradient:

0 min: 10% B 2.0 min: 10% B 15 min: 100% B 17 min: 100% B 17.05 min: 10% B 20 min: 10% B Solvent A is Water+0.1% Formic Acid Solvent B is Acetonitrile+0.1% Formic Acid

MS conditions: MS operates in switching mode (switching between positive and negative), scanning from 150 to 1500 amu.

Xray Powder Diffraction (XRPD) Method

Approximately 2 mg of sample was gently compressed on the XRPD zero back ground single obliquely cut silica sample holder. The sample was then loaded into a Philips X-Pert MPD diffractometer and analysed using the following experimental conditions:

Tube anode: Cu Generator tension: 40 kV Tube current: 40 mA Wavelength alpha1: 1.5406 Å Wavelength alpha2: 1.5444 Å Start angle [2 theta]: 5 End angle [2 theta]: 50 Continuous scan

In Vitro Replicon Assay for Assessment of HCV Antiviral Activity

Antiviral efficacy against genotype 1 HCV may be tested as follows: One day before addition of the test article, Huh5.2 cells, containing the HCV genotype 1b 1389luc-ubi-neo/NS3-3′/5.1 replicon (Vrolijk et al., 2003) and subcultured in cell growth medium [DMEM (Cat No. 41965039) supplemented with 10% FCS, 1% non-essential amino acids (11140035), 1% penicillin/streptomycin (15140148) and 2% Geneticin (10131027); Invitrogen] at a ratio of 1.3-1.4 and grown for 3-4 days in 75 cm² tissue culture flasks (Techno Plastic Products), were harvested and seeded in assay medium (DMEM, 10% FCS, 1% non-essential amino acids, 1% penicillin/streptomycin) at a density of 6 500 cells/well (100 μL/well) in 96-well tissue culture microtitre plates (Falcon, Beckton Dickinson for evaluation of the anti-metabolic effect and CulturPlate, Perkin Elmer for evaluation of antiviral effect). The microtitre plates are incubated overnight (37° C., 5% CO₂, 95-99% relative humidity), yielding a non-confluent cell monolayer.

Dilution series are prepared; each dilution series is performed in at least duplicate. Following assay setup, the microtitre plates are incubated for 72 hours (37° C., 5% CO₂, 95-99% relative humidity).

For the evaluation of anti-metabolic effects, the assay medium is aspirated, replaced with 75 μL of a 5% MTS (Promega) solution in phenol red-free medium and incubated for 1.5 hours (37° C., 5% CO₂, 95-99% relative humidity). Absorbance is measured at a wavelength of 498 nm (Safire², Tecan) and optical densities (OD values) are converted to percentage of untreated controls.

For the evaluation of antiviral effects, assay medium is aspirated and the cell monolayers are washed with PBS. The wash buffer is aspirated, 25 μL of Glo Lysis Buffer (Cat. No. E2661, Promega) is added after which lysis is allowed to proceed for 5 min at room temperature. Subsequently, 50 μL of Luciferase Assay System (Cat. No. E1501, Promega) is added and the luciferase luminescence signal is quantified immediately (1000 ms integration time/well, Safire², Tecan). Relative luminescence units are converted to percentage of untreated controls.

The EC50 and EC90 (values derived from the dose-response curve) represent the concentrations at which respectively 50% and 90% inhibition of viral replication would be observed. The CC50 (value derived from the dose-response curve) represents the concentration at which the metabolic activity of the cells would be reduced to 50% of the metabolic activity of untreated cells. The selectivity index (SI), indicative of the therapeutic window of the compound, is calculated as CC₅₀/EC₅₀.

A concentration of compound is considered to elicit a genuine antiviral effect in the HCV replicon system when, at that particular concentration, the anti-replicon effect is above the 70% threshold and no more than 30% reduction in metabolic activity is observed.

In Vitro Replicon Assay for Assessment of HCV Antiviral Activity in Genotypes La and 2a

The replicon cells (subgenomic replicons of genotype 1a (H77) and 2a (JFH-1)) are grown in Dulbecco's modified essential media (DMEM), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep), 1% glutamine, 1% non-essential amino acids, 250 μg/ml G418 in a 5% CO₂ incubator at 37° C. All cell culture reagents may be purchased from Mediatech (Herndon, Va.).

The replicon cells are trypsinized and seeded at 5×10³ cells per well in 96-well plates with the above media without G418. On the following day, the culture medium is replaced with DMEM containing compounds serially diluted in the presence of 5% FBS. The HCV replicon antiviral assay examines the effects of compounds in a serial of compound dilutions. Briefly, the cells containing the HCV replicon are seeded into 96-well plates. Test article is serially diluted with DMEM plus 5% FBS. The diluted compound is applied to appropriate wells in the plate. After 72 hr incubation at 37° C., the cells are processed. The intracellular RNA from each well is extracted with an RNeasy 96 kit (Qiagen). The level of HCV RNA is determined by a reverse transcriptase-real time PCR assay using TaqMan® One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, Calif.) and an ABI Prism 7900 sequence detection system (Applied Biosystems) a as described previously (Vrolijk et al., 2003). The cytotoxic effects are measured with TaqMan0 Ribosomal RNA Control Reagents (Applied Biosystems) as an indication of cell numbers. The amount of the HCV RNA and ribosomal RNA is then used to derive applicable IC₅₀ values (concentration inhibiting on replicon replication by 50%).

Assessment of Microsome Metabolisrn (Microsome Stability Assay)

Rate of metabolism in microsomes may be tested as follows:

Mouse or human liver microsomes were diluted with buffer C (0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4) to a concentration of 2.5 mg/mL. Microsomal stability samples were then prepared by adding 50 μL of 5 μM compound spiking solution (0.5 μL 10 mM DMSO stock solution in 9.5 μL ACN, added to 990 μL Buffer C) to 50 μL of microsomal solution (2.5 mg/mL), 110 μL Buffer C and mixed well. All samples were pre-incubated for approximately 15 minutes at 37° C. Following this, the reaction was initiated by adding 40 μL of the NADPH solution (12.5 mM) with gentle mixing. Aliquots (40 μL) were removed at 0, 215, 30, 45 and 60 minutes and quenched with ACN containing internal standard (120 μL). Protein was removed by centrifugation (4000 rpm, 15 min) and the sample plate analysed for compound concentration by LC-MS/MS. Half-lives were then calculated by standard methods, comparing the concentration of analyte with the amount originally present.

Assessment of Hepatocyte Stability

Cryopreserved hepatocytes, previously stored in liquid nitrogen are placed in a 37±1° C. shaking water bath for 2 min±15 sec. The hepatocytes are then added to 10× volume of pre-warmed Krebs-Henseleit bicarbonate (KHB) buffer (2000 mg/L glucose, no calcium carbonate and sodium bicarbonate, Sigma), mixed gently and centrifuged at 500 rpm for 3 minutes. After centrifugation, the supernatant is carefully removed and a 10× volume of pre-warmed KHB buffer added to resuspend the cell pellet. This is mixed gently and centrifuged at 500 rpm for 3 minutes. The supernatant is then removed and discarded. The cell viability and yield are then determined by cell counts, and these values used to generate human hepatocyte suspensions to the appropriate seeding density (viable cell density=2×10⁶ cells/mL). A 2× dosing solution is prepared in pre-warmed KHB (1% DMSO) (200 μM spiking solution: 20 μL of substrate stock solution (10 mM) in 980 μL of DMSO, 2× dosing solution: 10 μL of 200 μM spiking solution in 990 μL of KHB (2 μM after dilution).

50 μL of pre-warmed 2× dosing solution is added to the wells and 50 μL of pre-warmed hepatocyte solution (2×106 cells/mL) added and timing started. The plate is then incubated at 37° C. 100 μL of acetonitrile containing internal standard is added to each the wells after completion of incubation time (0, 15, 30, 60 and 120 minutes) mixed gently, and 50 μL of pre-warmed hepatocyte solution added (2×106 cells/mL). At the end of the incubation, cell viability is determined. Samples are centrifuged at 4000 rpm for 15 minutes at 4° C., supernatants diluted 2-fold with ultrapure water and compound levels analysed by LC-MS/MS.

Assessment of Water Solubility

Water solubility may be tested as follows: A 10 mM stock solution of the sanglifehrin analogue is prepared in 100% DMSO at room temperature. Triplicate 0.01 mL aliquots are made up to 0.5 mL with either 0.1 M PBS, pH 7.3 solution or 100% DMSO in amber vials. The resulting 0.2 mM solutions are shaken, at room temperature on an IKA® vibrax VXR shaker for 6 h, followed by transfer of the resulting solutions or suspensions into 2 mL Eppendorf tubes and centrifugation for 30 min at 13200 rpm. Aliquots of the supernatant fluid are then analysed by the LCMS method as described above.

Alternatively, solubility in PBS at pH7.4 may be tested as follows: A calibration curve is generated by diluting the test compounds and control compounds to 40 μM, 16 μM, 4 μM, 1.6 μM, 0.4 μM, 0.16 μM, 0.04 μM and 0.002 μM, with 50% MeOH in H₂O. The standard points are then further diluted 1:20 in MeOH:PBS 1:1. The final concentrations after 1:20 dilution are 2000 nM, 800 nM, 200 nM, 80 nM, 20 nM, 8 nM, 2 nM and 1 nM. Standards are then mixed with the same volume (1:1) of ACN containing internal standard (hydroxymacrocycle, 6). The samples are centrifuged (5 min, 12000 rpm), then analysed by LC/MS.

MeOH/ MeOH/ H₂O Working buffer Final Solution (1:1) solution Solution (1:1) solution (μL) (μL) (μM) (μL) (μL) (nM) 10 mM 10 240 → 400 400 μM 50 450 → 40 20 380 → 2000 20 480 → 16 20 380 → 800 40 μM 50 450 → 4 20 380 → 200 16 μM 50 450 → 1.6 20 380 → 80 4 μM 50 450 → 0.4 20 380 → 20 1.6 μM 50 450 → 0.16 20 380 → 8 0.4 μM 50 450 → 0.04 20 380 → 2 0.04 μM 50 950 → 0.002 20 380 → 1

Test compounds are prepared as stock solutions in DMSO at 10 mM concentration. The stock solutions are diluted in duplicate into PBS, pH7.4 in 1.5 mL Eppendorf tubes to a target concentration of 100 μM with a final DMSO concentration of 1% (e.g. 4 μL of 10 mM DMSO stock solution into 396 μL 100 mM phosphate buffer). Sample tubes are then gently shaken for 4 hours at room temperature. Samples are centrifuged (10 min, 15000 rpm) to precipitate undissolved particles. Supernatants are transferred into new tubes and diluted (the dilution factor for the individual test article is confirmed by the signal level of the compound on the applied analytical instrument) with PBS. Diluted samples are then mixed with the same volume (1:1) of MeOH. Samples are finally mixed with the same volume (1:1) of ACN containing internal standard (hydroxymacrocycle, 6) for LC-MS/MS analysis.

Assessment of Cell Permeability

Cell permeability may be tested as follows: The test compound is dissolved to 10 mM in DMSO and then diluted further in buffer to produce a final 10 μM dosing concentration. The fluorescence marker lucifer yellow is also included to monitor membrane integrity. Test compound is then applied to the apical surface of Caco-2 cell monolayers and compound permeation into the basolateral compartment is measured. This is performed in the reverse direction (basolateral to apical) to investigate active transport. LC-MS/MS is used to quantify levels of both the test and standard control compounds (such as Propanolol and Acebutolol).

In Vivo Assessment of Pharmacokinetics

In vivo assays may also be used to measure the bioavailability of a compound. Generally, a compound is administered to a test animal (e.g. mouse or rat) both intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time. The time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below.

Mice are dosed with 1, 10, or 100 mg/kg of the compound of the invention or the parent compound i.v. or p.o. Blood samples are taken at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420 and 2880 minutes and the concentration of the compound of the invention or parent compound in the sample is determined via HPLC. The time-course of plasma concentrations can then be used to derive key parameters such as the area under the plasma concentration-time curve (AUC—which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma drug concentration, the time at which maximum plasma drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half life, total body clearance, steady-state volume of distribution and F %. These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Egorin et al. 2002, and references therein.

In Vivo Assessment of Oral and Intravenous Pharmacokinetics (Specific Method)

For sanglifehrin analogues, whole blood is analysed. Compounds are formulated in 5% ethanol/5% cremophor EL/90% saline for both p.o. and i.v. administration. Groups of 3 male CD1 mice are dosed with either 1 mg/kg i.v. or 5 or 10 mg/kg p.o. Blood samples (40 μL) are taken via saphenous vein, pre-dose and at 0.25, 0.5, 2, 8, and 24 hours, and diluted with an equal amount of dH₂0 and put on dry ice immediately. Samples are stored at −70° C. until analysis. The concentration of the compound of the invention or parent compound in the sample is determined via LCMS as follows: 20 μL of blood:H₂O (1:1, v/v)/PK sample is added with 20 μL Internal standard (hydroxyl macrocycle, 6) at 100 ng/mL, 20 μL working solution/MeOH and 150 μL of ACN, vortexed for 1 minute at 1500 rpm, and centrifuged at 12000 rpm for 5 min. The supernatant is then injected into LC-MS/MS. The time-course of blood concentrations is plotted and used to derive area under the whole blood concentration-time curve (AUC—which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation). These values are used to generate PK parameters where possible.

In Vitro Assessment of Cytotoxicity

Huh-7 and HepG2 cells obtained from ATCC are grown in Dulbecco's modified essential media (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep) and 1% glutamine; whereas CEM cells (human T-cell leukemia cells obtained from ATCC) are grown in RPMI 1640 medium with 10% FBS, 1% pen-strep and 1% glutamine. Fresh human PBMCs are isolated from whole blood obtained from at least two normal screened donors. Briefly, peripheral blood cells are pelleted/washed 2-3 times by low speed centrifugation and resuspension in PBS to remove contaminating platelets. The washed blood cells are then diluted 1:1 with Dulbecco's phosphate buffered saline (D-PBS) and layered over 14 mL of Lymphocyte Separation Medium (LSM; cellgrow by Mediatech, Inc.; density 1.078+/−0.002 g/ml; Cat.#85-072-CL) in a 50 mL centrifuge tube and centrifuged for 30 minutes at 600×g. Banded PBMCs are gently aspirated from the resulting interface and subsequently washed 2× with PBS by low speed centrifugation. After the final wash, cells are counted by trypan blue exclusion and resuspended at 1×10⁷ cells/mL in RPMI 1640 supplemented with 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P. The cells are allowed to incubate for 48-72 hours at 37° C. After incubation, PBMCs are centrifuged and resuspended in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL gentamycin, and 20 U/mL recombinant human IL-2.

Compound cytotoxicity is evaluated by testing half-log concentrations of each compound in triplicate against the cells described above. Cell containing medium alone served as the cell control (CC). Huh-7 and HepG2 cells are seeded in 96-well plates at a concentration of 5×10³ cells per well. On the following day, the media is aspirated, and 100 μL of corresponding media containing 5% FBS is added. Test drug dilutions are prepared at a 2× concentration in microtiter tubes and 100 μL of each concentration is placed in appropriate wells in a standard format. After 72 hours, the cells are processed for cytotoxicity assessment. PBMCs are diluted in fresh medium and plated in the interior wells of a 96 well round bottom microplate at 5×10⁴ cells/well in a volume of 100 L. Similarly, CEM cells are plated at 1×10⁴ cells/well. Then, 100 μL of 2× preparations of the test drugs are added in appropriate wells in a standard format. The cultures are maintained for six to seven days and then processed for cytotoxicity determination.

Cytotoxicity is determined using CytoTox-ONE™ homogeneous membrane integrity assay kit (Promega). The assay measures the release of lactate dehyrodgenase (LDH) from cells with damaged membranes in a fluorometric, homogeneous format. LDH released into the culture medium is measured with a coupled enzymatic assay that results in the conversion of resazurin into a fluorescent resorufin product. The amount of fluorescence produced is proportional to the number of lysed cells. Six serially diluted concentrations of each compound are applied to the cells to derive where applicable TC50 (toxic concentration of the drug decreasing cell viability by 50%) and TC90 (toxic concentration of the drug decreasing cell viability by 90%) values.

In Vitro Assessment of Inhibition of MDR1 and MRP2 Transporters

To assess the inhibition and activation of the MDR1 (P-glycoprotein 1) and MRP2 transporters, an in vitro ATPase assay from Solvo Biotechnology Inc. can be used (Glavinas et al., 2003). The compounds (at 0.1, 1, 10 and 100 μM) are incubated with MDR1 or MRP2 membrane vesicles both in the absence and presence of vanadate to study the potential ATPase activation. In addition, similar incubations are conducted in the presence of verapamil/sulfasalazine in order to detect possible inhibition of the transporter ATPase activity. ATPase activity is measured by quantifying inorganic phosphate spectrophotometrically. Activation is calculated from the vanadate sensitive increase in ATPase activity. Inhibition is determined by decrease in verapamil/sulfasalazine mediated ATPase activity.

In Vitro Assessment of Inhibition of Pgp Transporters Using Mdck Cells

To assess the inhibition of the P-glycoprotein (Pgp/MDR1) transporter, an in vitro ATPase assay from Cyprotex was used. MDR1—MDCK cells obtained from the NIH (Rockville, Md., USA) were used. Following culture, the monolayers were prepared by rinsing both basolateral and apical surfaces twice with buffer at pH 7.4 and 37° C. Cells were then incubated with pH 7.4 buffer in both apical and basolateral compartments for 40 min at 37° C. and 5% CO₂ with a relative humidity of 95% to stabilise physiological parameters. For the apical to basolateral study (A-B), buffer at pH 7.4 was removed from the apical compartment and replaced with loperamide dosing solutions before being placed in the ‘companion’ plates. The solutions were prepared by diluting loperamide in DMSO with buffer to give a final loperamide concentration of 5 μM (final DMSO concentration adjusted to 1%). The fluorescent integrity marker Lucifer yellow was also included in the dosing solution. The experiment was performed in the presence and absence of the test compound (applied to both the apical and basolateral compartments). For basolateral to apical (B-A) study, the P-glycoprotein substrate, loperamide (final concentration=5 μM) was placed in the basolateral compartment. The experiment was performed in the presence and absence of the test compound (applied to the apical and basolateral compartments). Incubations were carried out in an atmosphere of 5% CO₂ with a relative humidity of 95% at 37° C. for 60 min. After the incubation period, the companion plate was removed and apical and basolateral samples diluted for analysis by LC-MS/MS. A single determination of each test compound concentration was performed. On each plate, a positive control inhibitor was also screened. The test compound was assessed at 0.1, 0.3, 1, 3, 10, 30 and 50 μM. The integrity of the monolayers throughout the experiment was checked by monitoring Lucifer yellow permeation using fluorimetric analysis. After analysis, an IC₅₀ was calculated (i.e., inhibitor concentration (test drug) achieving half maximal inhibition effect).

In Vitro Assessment of Inhibition of Uptake Transporters

To assess the inhibition of the OAT1B1 and OAT1B3 uptake transporters, an in vitro uptake transporter assay from Solvo Biotechnology Inc. was used. Uptake experiments with Test Article (TA) at 0.068, 0.2, 0.62, 1.8, 5.5, 16.7 and 50 μM, were performed on CHO cells stably expressing human SLC transporters OATP1B1 and OATP1B3. Parental cell line CHO-K was used as negative control. Cells (1×10⁵ in 200 μl 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 DMEM (F-12, Lonza, N.J., US) supplemented with 5 mM sodium butyrate) were plated on standard 96-well tissue culture plates and incubated 24 hours before the experiment at 37° C. in an atmosphere of 5% CO2 and 95% air. Before experiments the medium was aspirated by vacuum suction, cells were washed with 2×100 μl of Krebs-Henseleit buffer pH 7.3 (prepared from Sigma chemicals, Sigma-Aldrich, St Louis, Mo.). Uptake experiments were carried out at 37° C. in 50 μl of Krebs-Henseleit buffer (pH 7.3) containing the probe substrate and the TA or solvent, respectively. The organic solvent concentration was equal in each well, and did not exceed 1% v/v. The probe substrate for the OATP1B1 assay was E3S (0.1 μM) and for the OATP1B3 assay was Fluo-3 (10 μM). The translocated amount of probe substrate was determined for each well in cpm. Relative activities were calculated from the equation:

Activity%=(A−B)/(C−D)×100

Where A=translocated amount of substrate in the presence of TA on transfected cells, B=translocated amount of substrate in the presence of TA on parental cells, C=translocated amount of substrate in the presence of solvent on transfected cells and D=translocated amount of substrate in the presence of solvent on parental cells. IC₅₀ was defined as the TA concentration needed to inhibit transport of the probe substrate by 50%. IC₅₀ was derived from the three-parameter logistic equation; a curve fitted onto the relative activity vs. TA concentration plot by non-linear regression.

In Vitro Assessment of Inhibition of Efflux Transporters

To assess the inhibition of the MRP2, MRP3 and BSEP efflux transporters, an in vitro vesicular transporter assay from Solvo Biotechnology Inc. was used. The Test Articles (TAs) (at 0.068, 0.2, 0.62, 1.8, 5.5, 16.7 and 50 μM) were incubated with efflux transporter membrane vesicles (Solvo Biotechnology Inc.) both in the absence and presence of 4 mM ATP to distinguish between transporter mediated uptake and passive diffusion of TA's into the vesicles. In the case of MRP2 and MRP3 transporters reactions were carried out in the presence of 2 mM glutathione. Reaction mixtures were preincubated for ten minutes at 37° C. Reactions were started by the addition of 25 μl of 12 mM MgATP (4 mM final concentration in assay) or assay buffer for background controls. Reactions were stopped by adding 200 μl of ice-cold washing buffer and immediately followed by filtration on glass fiber filters in a 96-well format (filter plate). Scintillation buffer was added to the washed and dried filter plate and scintillation was counted subsequently. Probe substrates were taurocholate (2 μM) for BSEP vesicles and E₂17βG (1 μM) for MRP2 and MRP3 vesicles. For all wells the translocated amount of the probe substrate was determined in cpm units. Relative activities were calculated with the following equation:

Activity %=(A−B)/(C−D)×100 Where A=translocated amount of substrate in the presence of TA and ATP, B=translocated amount of substrate in the presence of TA, C=translocated amount of substrate in the presence of solvent and ATP and D=translocated amount of substrate in the presence of solvent. IC₅₀ was defined as the TA concentration needed to inhibit transport of the probe substrate by 50%. IC₅₀ was derived from the three-parameter logistic equation; a curve fitted onto the relative activity vs. TA concentration plot by non-linear regression.

In Vitro Assay for Assessment of HIV Antiviral Activity

Antiviral efficacy against HIV may be tested as follows: Blood derived CD4+ T-lymphocytes and macrophages are isolated as described previously (Bobardt et al., 2008). Briefly, human PBMCs were purified from fresh blood by banding on Ficoll-Hypaque (30 min, 800 g, 25° C.). Primary human CD4+ T cells were purified from PBMCs by positive selection with anti-CD4 Dynabeads and subsequent release using Detachabead. Cells were cultured in RPMI medium 1640 (Invitrogen) supplemented with 10% FCS, MEM amino acids, L-glutamine, MEM vitamins, sodium pyruvate, and penicillin plus streptomycin and were subsequently activated with bacterial superantigen staphylococcal enterotoxin B (SEB; 100 ng/ml) and mitomycin C-killed PBMC from another donor (10:1 PBMC:CD4 cell ratio). Three days after stimulation, cells were split 1:2 in medium containing IL-2 (200 units/ml final concentration). Cultures were then split 1:2 every 2 days in IL-2 medium and infected with HIV at 7 days after stimulation. For generating primary human macrophages, monocytes were purified from human PBMCs by negative selection and activated and cultured at a cell concentration of 106/ml in DMEM, supplemented with 10% FCS, MEM amino acids, L-glutamine, MEM vitamins, sodium pyruvate, and penicillin (100 units/ml), streptomycin (100 mg/ml), and 50 ng/ml recombinant human granulocyte—macrophage colony-stimulating factor (GM-CSF) and maintained at 37° C. in a humidified atmosphere supplemented with 5% CO₂. To obtain monocyte-derived macrophages, cells were allowed to adhere to plastic and cultured for 6 days to allow differentiation.

CD4+ HeLa cells, Jurkat cells, activated CD4+ peripheral blood T-lymphocytes and macrophages (500,000 cells/100 μL) were incubated with pNL4.3-GFP (X4 virus) or pNL4.3-BaL-GFP (R5 virus) (100 ng of p24) in the presence of increasing concentrations of test article, Forty-eight hours later, infection was scored by analyzing the percentage of GFP-positive cells by FACS and EC₅₀ calculated.

In Vitro Assay for Assessment of HBV Antiviral Activity

Antiviral efficacy against HBV may be tested as follows: HepG2 2.2.15 cells are plated in 96-well microtiter plates. After 16-24 hours the confluent monolayer of HepG2 2.2.15 cells is washed and the medium is replaced with complete medium containing various concentrations of a test compound in triplicate (e.g. six half-log concentrations). Three days later the culture medium is replaced with fresh medium containing the appropriately diluted test compounds. Six days following the initial administration of the test compound, the cell culture supernatant is collected, treated with pronase and then used in a real-time quantitative TaqMan qPCR assay. The PCR-amplified HBV DNA is detected in real-time by monitoring increases in fluorescence signals that result from the exonucleolytic degradation of a quenched fluorescent probe molecule that hybridizes to the amplified HBV DNA. For each PCR amplification, a standard curve is simultaneously generated using dilutions of purified HBV DNA. Antiviral activity is calculated from the reduction in HBV DNA levels (IC₅₀). A dye uptake assay is then employed to measure cell viability, which is used to calculate toxicity (TC₅₀). The therapeutic index (TI) is calculated as TC₅₀/IC₅₀.

In Vitro Mixed Lymphocyte Reaction (MLR) Assay for Assessment of Immunosuppressant Activity

Immunosuppressant activity was tested as follows: Peripheral blood mononuclear cell (PBMC) populations were purified from the blood of two normal, unrelated volunteer donors (A & B), using centrifugation over histopaque. Cells were counted and plated out at 1×10⁵ cells per well in 96 well plates in RPMI media, with supplements and 2% Human AB serum.

Culture conditions included: cell populations A & B alone and a mixed population of cells A&B in the absence or presence of test compounds, each at 6 different concentrations. Compounds were tested at doses ranging from 10 μM to 0.0001 μM in 1-log increments. Control wells contained a comparable concentration of vehicle (0.5% DMSO) to that present in the test compound wells. Cultures were established in triplicate in a 96 well plate and incubated at 37° C. in 5% CO₂ in a humidified atmosphere. 3H-thymidine was added on day 6 after assay set up and harvested 24 hrs later. The levels of proliferation between the different culture conditions were then compared.

The ability of each dilution of test compound to inhibit proliferation in the MLR was calculated as percentage inhibition. This allowed estimation of the IC₅₀ (concentration of test compound which resulted in a 50% reduction of counts per minute). In order to calculate the IC₅₀, the X axis was transformed to a log scale. Non-linear regression was used to fit to the mean data points. A sigmoidal variable slope was selected.

ELISA Analysis of Cyp-NS5A Interaction.

This assay was used to measure the disruption of Cyp-NS5A complexes, which can be used to show the potency of interaction with Cyclophilin D. Briefly, production and purification of recombinant GST, GST-CypD and Con1 NS5A-His proteins was carried out as described previously (Chatterji et al., 2010). Nunc MaxiSorb 8-well strip plates were coated with GST or GST-CypD for 16 h at 4° C. and blocked. Recombinant NS5A-His (1 ng/mL) was added to wells in 50 μL of binding buffer (20 mM Tris pH 7.9, 0.5 M NaCl, 10% glycerol, 10 mM DTT and 1% NP-40) for 16 h at 4° C. Captured NS5A-His was subsequently detected using mouse anti-His antibodies (1 μg/mL) (anti-6×His, Clontech) and rabbit anti-mouse-horseradish peroxidase phosphatase (HRP) antibodies (1:1000 dilution). All experiments were conducted twice using two different batches of recombinant CypD and NS5A proteins.

Anti-PPIAse Analysis of Cyclophilin Inhibition

An alternative methodology for analysing interaction with cyclophilins is described as follows: The PPlase activity of recombinant CypA or D, produced by thrombin cleavage of GST-CypA or D, was determined by following the rate of hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymotrypsin. Chymotrypsin only hydrolyzes the trans form of the peptide, and hydrolysis of the cis form, the concentration of which is maximized by using a stock dissolved in trifluoroethanol containing 470 mM LiCl, is limited by the rate of cis-trans isomerization. CypA or D was equilibrated for 1 h at 5° C. with selected test article using a drug concentration range from 0.1 to 20 nM. The reaction was started by addition of the peptide, and the change in absorbance was monitored spectrophotometrically at 10 data points per second. The blank rates of hydrolysis (in the absence of CypA or D) were subtracted from the rates in the presence of CypA or D. The initial rates of the enzymatic reaction were analyzed by first-order regression analysis of the time course of the change in absorbance.

EXAMPLES Example 1 Construction of an sfaA Deletion Mutant of Streptomyces sp. A92-308110 (DSM9954)

1.1 Construction of the sfaA Deletion Construct

The ˜7 kb EcoRV-Stul fragment of cosmid TL3006 (SEQ ID NO. 3) encompassing sfaA (nucleotide position 14396-21362, NCBI sequence accession number FJ809786) was excised by digestion with EcoRV and StuI and the resulting isolated fragment ligated directly into pKC1139 that had previously been digested with EcoRV and treated with shrimp alkaline phosphatase (Roche). This plasmid was designated pSGK268.

An in frame deletion of the sfaA gene contained within this clone was performed using the Red/ET recombination kit supplied by Gene Bridges (catalog number K006).

SfaA17161f (SEQ ID NO. 1) 5′-CGCTCTGTGGCGCCTGGTTTCCAAGCGGCTCGCGGACCGGCACCGGC ACATGCATAATTAACCCTCACTAAAGGGCG-3′ SfaA17825r (SEQ ID NO. 2) 5′-TGGATGTATCGTCGCAGGACGCCCAGAATTCACCTGCGACGTCCTCC AGATGCATTAATACGACTCACTATAGGGCTC-3′

Two oligonucleotides, SfaA17161f and SfaA17825r were used to amplify the neomycin marker from the FRT-PGK-gb2-neo-FRT template DNA supplied in the kit using KOD DNA polymerase. The resulting ˜1.7 kb amplified product was isolated by gel electrophoresis and purified from the gel with QiaEX resin.

Plasmid pSGK268 was transformed into E. coli DH10B using standard techniques and selected on plates containing apramycin (50 μg/ml). Introduction of the deletion construct was performed essentially following the Gene Bridges kit protocol. A single colony was grown overnight in 2TY apramycin (50 μg/ml) and transformed with the pRedET (tet) plasmid and selected on apramycin (50 μg/ml) and tetracycline (3 μg/ml) at 30° C. A single colony was used to prepare an overnight culture of this strain in 3 ml 2TY apramycin (50 μg/ml) and tetracycline (3 μg/ml) at 30 C. 0.5 ml of this culture was used to inoculate 10 ml 2TY apramycin (50 μg/ml) and tetracycline (3 μg/ml) at 30° C. and grown to an OD_(600nm) ˜0.5. 1.4 ml of this culture was transferred to each of 2 eppendorf tubes and 50 ul 10 arabinose added to one tube to induce expression of the Red/ET recombination proteins. Tubes were shaken for ˜1 hour at 37° C. Induced and non-induced cells were pelleted in a bench top centrifuge and washed twice with chilled sterile water; resuspending and centrifuging to pellet the cells each time. The resulting pellets were suspended in about 30-40 μl of water and kept on ice. The 1.7 kb disruption fragment isolated previously was added to the induced and non-induced tubes and transferred to 1 mm Biorad electrocuvettes on ice. The samples were electroporated (Biorad Micropulser at 1.8 kV, resulting time constant ˜4 ms) and 1 ml 2TY (no antibiotics) added and mixed to remove the cells from the cuvette. Cells were incubated for ˜3 hours at 37° C. with shaking (1100 rpm, eppendorf thermomixer compact) before plating onto 2TY plates containing apramycin (50 μg/ml and kanamycin 25 μg/ml and incubating over night at 37° C. Colonies from the induced sample plates were streaked onto 2TY plates containing kanamycin at 50 μg/ml to purify and confirm introduction of the kanamycin resistance cassette. PCR on individual bacterial colonies was used to confirm the introduction of the cassette. Plasmids were prepared from these cultures and digested to confirm the expected plasmid pSGK270. Plasmids were then digested with NsiI to remove the marker fragment, and the remainder religated to produce the sfaA in-frame deletion construct pSGK271.

1.2 Conjugation of Streptomyces sp. A92-308110 (DSM9954) and Introduction of an sfaA Deletion

Plasmid pSGK271 was transformed into E. coli ET12567 pUZ8002 using standard techniques and selected on 2TY plates containing apramycin (50 μg/ml), kanamycin (25 μg/ml) and chloroamphenicol (10 μg/ml). The resulting strain was inoculated into 3 ml liquid 2TY containing apramycin (50 μg/ml), kanamycin (25 μg/ml) and chloroamphenicol (10 μg/ml) and incubated overnight at 37° C., 250 rpm. 0.8 ml of this culture was used to inoculate 10 ml liquid 2TY containing apramycin (50 μg/ml), kanamycin (25 μg/ml) and chloroamphenicol (10 μg/ml) in a 50 ml Falcon tube and incubated at 37° C. 250 rpm until OD_(600 nm) ˜0.5 was reached. The resulting culture was centrifuged at 3500 rpm for 10 minutes at 4° C., washed twice with 10 ml 2TY media using centrifugation to pellet the cells after each wash. The resulting pellet was resuspended in 0.5 ml 2TY and kept on ice before use. This process was timed to coincide with the complete preparation of Streptomyces spores described below.

Spores of Streptomyces sp. A92-308110 (DSM9954) (Biot-4370) were harvested from a 1-2 week old confluent plate by resuspending in ˜3 ml 20% glycerol. Spores were centrifuged (5000 rpm, 10 minutes room temperature) and washed twice with 50 mM TES buffer before resuspending in 1 ml 50 mM TES buffer and splitting between 2 eppendorf tubes. These tubes were heat shocked at 50° C. for 10 minutes in a water bath before adding 0.5 ml 2TY and incubating in an Eppendorf Thermomixer compact at 37° C. for 4-5 hours.

The prepared E. coli ET12567 pUZ8002 pSGK271 and Biot-4370 were mixed at ratios 1:1 (250 μL each strain) and 1:3 (100 μL E. coli) and immediately spread on R6 plates and transferred to a 37° C. incubator. After approximately 2 hours incubation these plates were overlaid with 2 ml of sterile water containing nalidixic acid to give a final in-plate concentration of 25 μg/L. Plates were returned to the 37° C. incubator overnight before overlaying with 2 ml of sterile water containing apramycin to give a final in-plate concentration of 20-25 μg/L. Ex-conjugant colonies appearing after ˜4-7 days were patched to ISP4 media containing apramycin (25 μg/L) and nalidixic acid (25 μg/L) and incubated at 37° C. Once adequate mycelial growth was observed strains were repatched to ISP4 media containing apramycin (25 μg/L) at 37° C. and allowed to sporulate. Strains were then subcultured three times (to promote removal of the temperature sensitive plasmid) by patching to ISP4 (without antibiotic) and incubating at 37° C. for 3-4 days. Strains were finally patched to ISP4 and incubated at 28° C. to allow full sporulation (5-7 days). Spores were harvested and serially diluted onto ISP4 plates at 28° C. to allow selection of single colonies. Sporulated single colonies were doubly patched to ISP4 plates with or without apramycin (25 μg/L) to confirm loss of plasmid and allowed to grow ˜7 days before testing for production of sanglifehrins.

1.3 Screening Strains for Production of Sanglifehrins in Falcon Tubes

A single ˜7 mm agar plug of a well sporulated strain was used to inoculate 7 ml of sterile SM25-3 media and incubated at 27° C. 200 rpm in a 2″ throw shaker. After 48 hours of growth 0.7 ml of this culture was transferred to a sterilised falcon tube containing 7 ml of SGP2 media with 5% HP20 resin. Cultures were grown at 24° C. 300 rpm on a 1 inch throw shaking incubator for 5 days before harvest. 0.8 ml bacterial culture was removed and aliquoted into a 2 ml eppendorf tube ensuring adequate dispersal of the resin in throughout the culture prior to aliquoting. 0.8 ml acetonitrile and 15 μl of formic acid were added and the tube mixed for about 30 minutes. The mixture was cleared by centrifugation and 170 μl of the extract removed into a HPLC vial and analysed by HPLC.

1.4 Analysis of Strains for Reversion to Wild Type or sfaA Phenotype.

Extracts of strains were analysed by HPLC. Strains that produced sanglifehrin A and B were not analysed further as these had reverted to wild type. Strains lacking sanglifehrin A and B production showed small levels (˜1-2 mg/L) of a peak retention time 6.5 minutes that displayed a sanglifehrin like chromophore. Analysis by LCMS indicated this peak had a m/z 1073, −16 units from the expected m/z of sanglifehrin. It was postulated this peak was due to incorporation of phenylalanine in absence of meta-hydroxytyrosine.

Eight strains showing loss of sanglifeherin production were subsequently regrown to assess whether the potential sfaA mutation could be complemented chemically allowing a mutasynthetic process to novel sanglifehrins. Strains were grown in SM25-3 seed media for 48 hours before transferring to SGP2 production media with 5% resin. After a further 24 hours growth strains were fed in triplicate with 2 mM DL meta-hydroxytyrosine (addition of 100 ul of a 0.16M solution in 1M HCL) or 2 mM L-phenylalanine with an unfed strain used as control. Strains were also fed pipecolic acid (2 mM) in methanol) to enhance product yields. Strains were harvested after a further 4 days growth and extracted and analysed by HPLC. Meta-hydroxy tyrosine was shown to completely complement the sfaA mutation and addition of L-phenylalanine increased levels of the −16 amu compound. Strain Biot-4585 was chosen for further study as the sfaA deletion mutant.

Example 2 Other Methods for Construction of the sfaA Deletion Construct

Other methods can be used to generate sfaA deletion mutants. Examples include sfaA insertional inactivation mutants (such as example 12 from WO2010/034243). This strain was generated as described in WO2010/034243, and given the strain designation BIOT-4452.

In an alternative procedure to generate the deletion of sfaA two oligonucleotides 15209F 5′-CAGAGAATTCGCGGTACGGGGCGGACGACAAGGTGTC-3′ (SEQ ID NO. 4) and 17219R 5′-GCGCATGCATGTGCCGGTGCCGGTCCGCGAGCCGCTTGG-3′ (SEQ ID NO. 5) are used to amplify an upstream region of homology using cosmid TL3006 (SEQ ID NO. 3) as template and KOD DNA polymerase. The amplified product is treated with T4 polynucleotide kinase (NEB) and cloned into pUC18 that has been dephosphorylated by treating with shrimp alkaline phosphatase (Roche). The resulting construct is checked by restriction digestion and thoroughly sequenced to ensure the desired sequence is generated and that errors have not been introduced during polymerase amplification. This construct is digested with EcoRI and NsiI and the products analysed by gel electrophoresis. The desired sequence-containing band (i.e. upstream homology ˜2 kb) is excised from the gel and purified using standard procedures (QiaEX resin). A second series of oligonucleotides: 17766F 5′-CCTCATGCATCTGGAGGACGTCGCAGGTGAATTCTGGGCG-3′ (SEQ ID NO. 6)

and 19763R 5′-GGGCAAGCTTCTCCTGGCTGAGCTTGAACATCG-3′ (SEQ ID NO. 7) are used to amplify a downstream region of homology using cosmid TL3006 (SEQ ID NO. 3) as template and KOD DNA polymerase. The amplified product is treated with T4 polynucleotide kinase (NEB) and cloned into pUC18 that has been dephosphorylated by treating with shrimp alkaline phosphatase (Roche). The resulting construct is analysed by restriction digestion and thoroughly sequenced to ensure the desired sequence is generated and that errors have not been introduced during polymerase amplification. This construct is digested with HindIII and NsiI and the products analysed by gel electrophoresis. The desired sequence-containing band (i.e. downstream homology ˜2 kb) is excised from the gel and purified using standard procedures (QiaEX resin). Vector pKC1139 is digested with EcoRI and HindIII and the large vector fragment isolated by gel electrophoresis and purified by standard methods (QiaEX resin). The isolated upstream and downstream homology fragments are then cloned into this fragment of pKC1139 in a three-way ligation to generate the desired sfaA deletion construct.

In a further alternative procedure for generation of a sfaA deletion construct commercial gene synthesis (i.e. Genscript or other vendor) is used to generate a construct containing the desired sequence (SEQ ID NO. 8). This purchased construct is digested using BamHI and XbaI to excise the sequence of interest and the products analysed by gel electrophoresis. The desired sequence-containing band (−4 kb) is excised from the gel and purified using standard procedures. Vector pKC1139 is digested with BamHI and XbaI and the large fragment isolated by gel electrophoresis and purified by standard methods. The two isolated fragments are then ligated together to generate the desired sfaA deletion construct.

These alternative sfaA deletion constructs are introduced into Streptomyces sp. A92-308110 (DSM9954) by conjugation and selection for the secondary cross using the methods described in Example 1.2. Growth and analysis of strains constructed in this way also follows the methods described in Example 1.2

Example 3 Array Feed of the sfaA Deletion Mutant

Spore stocks of a mutant disrupted in sfaA (BIOT-4452 or BIOT-4585) were prepared after growth on MAM, ISP4, ISP3 or ISP2 medium, and preserved in 20% w/v glycerol in distilled water and stored at −80° C. Vegetative cultures (seed cultures) were prepared by inoculating spore stock (1% v/v) into 7 mL seed medium (SM25 medium) in 50 mL centrifuge tubes with foam plugs. The culture tubes were incubated at 27° C., 250 rpm (5 cm throw) for 48 h. From the seed culture 10% (v/v) was transferred into 7 mL production medium SGP-2 in 50 mL centrifuge tubes with foam plugs. Cultivation was carried out at 24° C. and 300 rpm (2.5 cm throw). For production of sanglifehrin mutasynthetic analogues, 0.05 mL of a 0.32 M solution (in 1N HCl) of the feed compound (mutasynthon) was added to each tube at 24 hours post inoculation to give a final concentration of 2 mM. Additionally, 0.05 ml of a 0.32 M solution of piperazic acid (in methanol) was added to each tube at 24 hours to give a final concentration of 2 mM. Cultivation was continued for an additional four days post feeding.

Samples were extracted by transferring 0.8 ml of the whole broth into a 2 ml capped eppendorf tube. 0.8 ml of acetonitrile was added, along with 0.015 ml of formic acid. The mixture was then shaken for 30 minutes on a vibrax. The tube was then centrifuged at 13000 rpm for 10 minutes and 0.15 ml of the supernatant was removed for analysis. Extracts were analysed as described in general methods.

Table 1 shows the mutasynthons that were fed in this way, along with the LCMS H+ and Na+ adducts, anticipated molecular mass and retention time of the sanglifehrin mutasynthetic products observed. The major peaks, relating to the sanglifehrin A analogues, are shown. In all cases, LCMS peaks were also seen for the sanglifehrin B analogues (Mass −18).

TABLE 1 [M − H]⁻ [M + Na]⁺ molecular retention mutasynthon observed observed mass time fed mutasynthon name (m/z) (m/z) (amu) (minutes)

2-amino-3-(3-fluoro-5- hydroxyphenyl)propanoic acid 1106.4 1130.4 1107.4 5.7

methyl 2-amino-3-(3- fluoro-5-hydroxyphenyl) proprionate 1106.4 1130.4 1107.4 5.7

Example 4 Isolation of 63-Fluoro Sanglifehrin A, Intermediate Compound 14

Fermentation carried out as described in general methods utilising methyl 2-amino-3-(3-fluoro-5-hydroxyphenyl)propanoate and DL-piperazic acid as precursors, both were added at 26 hours.

After harvesting the culture broths were pooled and adjusted to approx. pH 3 with formic acid and centrifuged (3300 g) for 25 mins to separate the cells and resin from the clarified broth. The clarified broth was discarded after assay having confirmed less than 5% of target compound present. The cells and resin were stirred with 2 volumes of acetonitrile for 1 hr using a magnetic stirrer. The acetonitrile extract was recovered either by centrifugation or by allowing it to settle under gravity. A second acetonitrile extraction of the cells and resin was then performed under the same conditions. The combined acetonitrile extracts were concentrated to a residual aqueous volume under reduced pressure and then adjusted to pH 6. This was extracted twice with ethyl acetate and the combined organics taken to dryness under reduced pressure to give the final crude (1.3 g).

The crude extract (1.3 g) was dissolved in ethyl acetate (2 ml) and loaded onto a silica gel column (10×2 cm) conditioned with ethyl acetate (500 ml). The column was eluted with ethyl acetate and then with stepwise increases in acetone (10%, 20%, 30%, etc. in ethyl acetate). Approx. 250 mL fractions were collected and the target compound identified by analytical LC, combined and taken to dryness. This material (278 mg) was dissolved in methanol (1.8 ml) and purified by preparative HPLC. A Waters Xterra MSC18 column (10 micron, 19 cm×250 mm) was used with solvent pumped at 21 mL/min. Solvent A was water and solvent B was acetonitrile. The column was run isocratically at 50% B for 6 minutes following the injection followed by a gradient to 100% B at 30 minutes. Pure fractions were identified by HPLC-UV and combined. These fractions were taken to dryness under reduced pressure to yield the target compound as an off-white amorphous solid (20 mg).

Example 5 Synthesis of diethyl (2-(1,2-oxazinan-2-yl)-2-oxoethyl)phosphonate

To a solution of 21-1 (ChemCollect, Germany)(100 mg, 0.81 mmol), Et₃N (246 mg, 2.43 mmol) in dry DCM (5 mL) was added dropwise chloroacetyl chloride (138 mg, 1.22 mmol). The reaction mixture was stirred at room temperature for 3 h, poured into ice water, and extracted with ethyl acetate. The organic layer was washed with brine and dried over Na₂SO₄, filtered, concentrated in vacuo. The residue (21-2) was used to the next step without any further purification. (123 mg, 90% yield).

A mixture of 21-2 (123 mg, 0.75 mmol) and triethyl phosphite (250 mg, 1.50 mmol) were stirred at 140° C. for 6 h. The reaction mixture was cooled to room temperature and was purified by flash chromatography to yield 21.

Alternative synthesis of synthesis of diethyl (2-(1,2-oxazinan-2-yl)-2-oxoethyl)phosphonate, 21

To a solution of t-BuOK (84.0 g, 0.75 mol) in tetrahydrofuran (2.0 L) was added 21a-1 (50.0 g, 0.38 mol) portion-wise at room temperature. the mixture was stirred for 1 h at room temperature. 1,4-Dibromobutane (81.2 g, 0.38 mol) was added dropwise at room temperature. Then the mixture was stirred at 80° C. for 16 h. After cooling down, water (2000 mL) was added, the mixture was extracted with ethyl acetate (2×1000 mL). The combined organic later was dried over anhydrous Na₂SO₄ for 16 h, after filtration and concentration, the residue was purified by silica-gel column chromatography (eluent: petroleum ether:ethyl acetate=100:1 to 10:1) to give 21a-2 (57 g) as a colorless oil.

General Procedure for Preparation of 21a-3

To a solution of 21a-2 (55 g, 0.29 mol) in tert-butyl methyl ether, TBME (80 mL) was added a solution of 4N HCl (600 ml, in TBME) at room temperature, the mixture was stirred for 3 h at room temperature. The precipitated solid was filtered and washed with TBME (50 mL) to give 21a-3 (30 g) as a white solid.

General Procedure for Preparation of 21

To a stirred solution of 21a-4 (35 g, 0.18 mol), hydroxybenzotriazole (HOBT) (29 g, 0.21 mol) and Et₃N (71 mL, 0.51 mol) in anhydrous dichloromethane (550 mL) was added 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (41 g, 0.21 mol) portion-wise at 0° C. The reaction mixture was stirred at 0° C. for 0.5 h, then 21a-3 (24 g, 0.20 mol) was added at 0° C. and stirred for 16 h. Then TLC (petroleum ether/ethyl acetate: 3/1) showed that the reaction was complete. At this time the reaction mixture was slowly poured into water (500 mL) with vigorous stirring. The mixture was extracted with dichloromethane (2×200 mL). The combined organic layer was washed with brine (2×100 mL), dried with Na₂SO₄, filtered and concentrated to afford crude product. Chromatography (petroleum ether/ethyl acetate, 100:1 to 10:1) gave 21 (38 g) as a yellow oil.

Example 6 Preparation of Intermediate Compound 23-3

To a stirred solution of 14 (430 mg, 0.38 mmol), (DHQ)₂PHAL (18.6 mg, 0.024 mmol), osmium tetroxide (0.156 mL, 0.012 mmol) in tert-butyl alcohol (2.5 wt %, 0.079 mmol/ml), and methanesulfonamide (74 mg, 0.77 mmol) in 20 mL tert-butyl alcohol were added at room temperature, a solution of potassium ferricyanide (382 mg, 1.16 mmol) and potassium carbonate (160 mg, 1.16 mmol) in 20 mL water, resulting in a brown emulsion. After 2 h a solution of sodium sulfite was added, and stirring was continued for 20 min. The resulting mixture was extracted with ethyl acetate (3×50 ml). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, purified by reverse-phase flash chromatography to yield 23-2 as a white solid.

To a stirred solution of 23-2 (240 mg, 0.21 mmol) in 24 mL of a 2:1 mixture of THF and water was added sodium periodate (91 mg, 0.42 mmol). The resulting mixture was stirred at room temperature for 3 h, and then saturated aqueous sodium bicarbonate was added. This mixture was extracted with three portions of ethyl acetate. The combined organic layers were washed with one portion of water and two portions of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography to yield 23-3.

Example 7 Preparation of Compound 24

To a solution of 21 (42 mg, 0.168 mmol) in THF (2.0 mL) was added NaH (1.2 mg, 0.05 mmol) in anhydrous THF (0.2 mL) at 0° C. with stirring. The solution was then stirred at 20° C. until it became clear. Then 23-3 (30 mg, 0.042 mmol) was added to the clear solution and the mixture stirred at 20° C. for 2 h. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (3×20 mL). The organic layer was washed with brine and dried over Na₂SO₄, filtered and reduced in vacuo. The residue was purified by preparative HPLC to obtained 24 as an amorphous white solid.

Example 8 Preparation of Compound 24 in Solid Crystalline Form (Form I)

10 mg of amorphous compound 24 was slurried in methyl isobutyl ketone (MIBK) (500 μL, 50 volumes) and then the temperature was cycled between ambient and 40° C. every 4 hours for a total of 5 days. The resulting solid was isolated by decanting off excess solvent followed by drying under vacuum to yield compound 24 in solid crystalline form (Form I). The XRPD pattern of Form I of compound 24 is illustrated in FIG. 2 and the peaks and their relative intensities are listed in Table 2 below. The method of obtaining the XRPD data is described in the General Methods.

TABLE 2 Peak No. Position [°2Theta] Relative Intensity [%] 1 6.2097 6.86 2 6.5031 7.76 3 8.2581 27.43 4 8.4838 33.64 5 9.5994 23.88 6 10.0981 8.54 7 11.0546 29.76 8 12.5883 14.81 9 13.1703 7.1 10 13.9184 100 11 14.2891 13.04 12 14.9759 10.37 13 15.3159 5.81 14 16.8844 18.15 15 17.1816 9.72 16 17.7384 53.03 17 18.1703 9.02 18 18.5613 32.19 19 19.0241 52.81 20 19.4201 5.08 21 20.0954 13.7 22 20.449 63.25 23 20.8962 43.44 24 21.1871 15.02 25 21.6388 16.08 26 23.0029 50.8 27 23.2869 17.19 28 23.6883 17.16 29 24.1071 13.7 30 24.2587 19.55 31 24.9948 13.34 32 25.209 26.16 33 25.9577 10.06 34 26.4298 9.38 35 27.3687 11.1 36 29.0171 7.95 37 29.5603 5.14 38 30.0609 7.35 39 30.5824 6.5 40 32.1814 4.39 41 32.6521 6.74 42 33.5957 6.6 43 34.7946 9.04

Example 9 Biological Data—HCV Replicon and Analysis

Compounds were analysed in the genotype 1b replicon assay using Huh5.2 cells as described in the General Methods. Cyclosporine A, 1, DEBIO-025, 2, sanglifehrin A, 5, and the hydroxymacrocycle, 6 were included as a comparison.

Selectivity index Compound EC50 (μM) CC50 (μM) (CC50/EC50) Cyclosporine A, 1 0.62 28 52 DEBIO-025, 2 0.096 11.2 111 Sanglifehrin A, 5 0.318 9.1 28.7 Hydroxymacrocycle, 6 8.4 83.6 9.9 24 0.033 >100 >3030

As can be seen, the compound of the invention, 24 is significantly more potent in the Huh5.2 replicon assay (as shown by the low EC₅₀), with significantly better selectivity against the cell line (as shown by a high selectivity index) as compared to CsA, Debio-025, SfA and the hydroxymacrocycle.

Example 10 Biological Data—Activity Against HIV

Compounds were analysed in an HIV antiviral assay using HeLa cells as described in the General Methods. Cyclosporine A, 1, DEBIO-025, 2, and the HIV antivirals emtricitabine and tenofovir were included as a comparison.

HeLa cells Compound EC₅₀ (μM) Cyclosporine A, 1 5.3 DEBIO-025, 2 1.5 Emtricitabine 0.4 Tenofovir 1.05 24 0.13

As can be seen, the compound of the invention, 24, is significantly more potent than CsA, DEBIO-025, emtricitabine and tenofovir at inhibiting HIV infection in this assay.

Example 11 Biological Data—Mouse In Vivo Oral and iv PK

To assess the pharmacokinetics of the compounds in an in vivo setting, compounds were dosed po at 10 or 5 mg/kg and iv at 1 mg/kg to groups of CD1 mice. Pharmacokinetic analysis was carried out as described in the general methods. The PK parameters are shown below.

Dose level Clearance po AUC_(last) Compound (mg/kg) (L/hr/kg) (ng * hr/mL) Sanglifehrin A, 5 10 0.054 2332 24 5 0.017 8223

As can be seen, compounds 24 has reduced clearance and increased oral exposure (as shown by a high po AUC_(last)), compared to sanglifehrin A.

Example 12 Biological Data—Inhibition of CypA PPlase Activity

To assess the direct inhibition of CypA Peptidyl Prolyl cis-trans Isomerase (PPlase) activity, a method was used as described in the general methods. Cyclosporine A, 1, DEBIO-025, 2 and Sanglifehrin A, 5 were included as controls.

CypA PPIase Compound IC₅₀ (nM) Cyclosporine A, 1 9.7 DEBIO-025, 2 0.8 Sanglifehrin A, 5 2.4 24 0.31

As can be seen, compound of the invention, 24, inhibits CypA PPlase activity more potently than Sanglifehrin A, DEBIO-025 and Cyclosporine A.

Example 13 Biological Data—Inhibition of Bilirubin Transporters

To assess the potential of off-target inhibition of bilirubin transporters, thought to be the reason for the dose-limiting hyperbilirubinaemia seen with DEBIO-025, in vitro analysis of transporter inhibition was carried out as described in the general methods.

OATP1B1 OATP1B3 IC₅₀ IC₅₀ MRP2 IC₅₀ MRP3 IC₅₀ Compound (μM) (μM) (μM) (μM) Cyclosporine A, 1 0.85 0.13 4.1 3.1 DEBIO-025, 2 0.45 0.19 16.0 >50 24 4.3 1.8 >50 >50

As can be seen, the compound of the invention, 24, shows much less inhibition of conjugated and unconjugated bilirubin transporters as compared to DEBIO-025 and Cyclosporine A.

Example 14 Biological Data—Inhibition of Xenobiotic Transporters

To assess the potential of Drug Drug Interactions (DDIs) via inhibition of xenobiotic transporters, in vitro analysis of P-glycoprotein (Pgp/MDR1) and Bile Salt Export Pump (BSEP) inhibition was carried out as described in the general methods.

Compound Pgp IC₅₀ (μM) BSEP IC₅₀ (μM) Cyclosporine A, 1 0.73 0.46 DEBIO-025, 2 0.72 0.18 24 >50 12.3

As can be seen, the compound of the invention, 24, shows much less inhibition of xenobiotic transporters, potentially involved in Drug-Drug Interactions, as compared to DEBIO-025 and Cyclosporine A.

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All references including patent and patent applications referred to in this application are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps. 

1. A compound of formula (I):

including any tautomer thereof; or an isomer thereof in which the C26, 27 C═C bond shown as trans is cis; and including a methanol adduct thereof in which a ketal is formed by the combination of the C-53 keto (if present) and the C-15 hydroxyl group and methanol; or a pharmaceutically acceptable salt thereof.
 2. A compound according to claim 1 is solid crystalline form.
 3. A compound according to claim 2 in the form of its Form I crystalline polymorph.
 4. A pharmaceutical composition comprising a compound according to claim 1 together with a pharmaceutically acceptable diluent or carrier.
 5. A pharmaceutical composition comprising a compound according to claim 1 together with a pharmaceutically acceptable diluent or carrier further comprising a second or subsequent active ingredient.
 6. A method of treatment of viral infections such as HCV or HIV infection or for use as an immunosuppressant or an anti-inflammatory agent which comprises administering to a subject a therapeutically effective amount of a compound according to claim
 1. 7. A process for preparing a compound according to claim 1 which comprises reacting a compound of formula (V)

wherein each R₁₁ is independently C₁₋₄ alkyl or benzyl; with an aldehydic macrocycle (compound of formula VI):


8. A compound of formula (VI):


9. A process for the preparation of a compound of formula (I) in the form of its Form I crystalline polymorph according to claim 3 comprising the step of crystallizing a compound of formula (I) from methyl isobutyl ketone. 